HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

PubMed Central

Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

2010-01-01

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

PubMed

Lai, J; Ma, S W; Zhu, R H; Rothman, R B; Lentes, K U; Porreca, F

1994-10-27

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

Osteomyelitis Treatment with Nanometer-Sized Hydroxyapatite Particles as a Delivery Vehicle for a Ciprofloxacin- Bisphosphonate Conjugate; New Fluoroquinolone-Bisphosphonate Derivatives Show Similar Binding Affinity to Hydroxyapatite and Improved Antibacterial Activity Against Drug-Resistant Pathogens

DTIC Science & Technology

2008-12-01

1 OSTEOMYELITIS TREATMENT WITH NANOMETER-SIZED HYDROXYAPATITE PARTICLES AS A DELIVERY VEHICLE FOR A CIPROFLOXACIN- BISPHOSPHONATE CONJUGATE; NEW...FLUOROQUINOLONE-BISPHOSPHONATE DERIVATIVES SHOW SIMILAR BINDING AFFINITY TO HYDROXYAPATITE AND IMPROVED ANTIBACTERIAL ACTIVITY AGAINST DRUG-RESISTANT...vivo OM model. Current studies contrast two CP homeostatic bone-substitute particles, nanometer-sized hydroxyapatite NanOss™ (Nan), and µ-sized

[125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

PubMed

Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

2002-07-01

The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor.

Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

PubMed

Rosenfeld, M R; Dvorkin, B; Klein, P N; Makman, M H

1982-03-04

Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases.

PubMed

Vermeiren, Céline; Motte, Philippe; Viot, Delphine; Mairet-Coello, Georges; Courade, Jean-Philippe; Citron, Martin; Mercier, Joël; Hannestad, Jonas; Gillard, Michel

2018-02-01

Lilly/Avid's AV-1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimer's disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [ 18 F]AV-1451 uptake in Alzheimer's disease may not be possible. The aim of the present study was to characterize the in vitro binding of AV-1451 to understand and identify potential off-target binding that could explain the poor discrimination observed in PSP patients. [ 3 H]AV-1451 and AV-1451 were characterized in in vitro binding assays using recombinant and native proteins/tissues from postmortem samples of controls and Alzheimer's disease and PSP patients. [ 3 H]AV-1451 binds to multiple sites with nanomolar affinities in brain homogenates and to tau fibrils isolated from Alzheimer's disease or PSP patients. [ 3 H]AV-1451 also binds with similarly high affinities in brain homogenates devoid of tau pathology. This unexpected binding was demonstrated to be because of nanomolar affinities of [ 3 H]AV-1451 for monoamine oxidase A and B enzymes. High affinity of AV-1451 for monoamine oxidase proteins may limit its utility as a tau PET tracer in PSP and Alzheimer's disease because of high levels of monoamine oxidase expression in brain regions also affected by tau deposition, especially if monoamine oxidase levels change over time or with a treatment intervention. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

A Single Glycine-Alanine Exchange Directs Ligand Specificity of the Elephant Progestin Receptor

PubMed Central

Wierer, Michael; Schrey, Anna K.; Kühne, Ronald; Ulbrich, Susanne E.

2012-01-01

The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems. PMID:23209719

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

PubMed

Tahara, A; Tsukada, J; Ishii, N; Tomura, Y; Wada, K; Kusayama, T; Yatsu, T; Uchida, W; Tanaka, A

1999-10-22

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.

Opioid agonists binding and responses in SH-SY5Y cells

NASA Technical Reports Server (NTRS)

Costa, E. M.; Hoffmann, B. B.; Loew, G. H.

1992-01-01

SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.

Influence of differentiation on muscarinic receptors in N1E 115 neuroblastoma cells.

PubMed

Buyse, M A; Lefebvre, R A; Fraeyman, N H

1989-01-01

The effect of inducing morphological differentiation in N1E 115 mouse neuroblastoma cells on the number of muscarinic receptors and the ligand binding affinity was investigated using the lipophylic quinuclidinyl benzylate and the hydrophylic N-methylscopolamine as tritiated ligands. Induction of morphological differentiation was accompanied by a two- to three-fold increase of the number of receptors when assayed in a broken cell preparation; the ligand binding affinity was unaffected by differentiation. Using intact cells, this increase was not paralleled by a similar increase in binding sites accessible for N-methylscopolamine, which binds preferentially to extracellular sites.

Characterization of the Binding of a Potent Synthetic Androgen, Methyltrienolone, to Human Tissues

PubMed Central

Menon, Mani; Tananis, Catherine E.; Hicks, L. Louise; Hawkins, Edward F.; McLoughlin, Martin G.; Walsh, Patrick C.

1978-01-01

The potent synthetic androgen methytrienolone (R 1881), which does not bind to serum proteins, was utilized to characterize binding to receptors in human androgen responsive tissues. Cytosol extracts prepared from hypertrophic prostates (BPH) were utilized as the source of receptor for the initial studies. High affinity binding was detected in the cytosol of 29 of 30 samples of BPH (average number of binding sites, 45.8±4.7 fmol/mg of protein; dissociation constant, 0.9±0.2 nM). This binding had the characteristics of a receptor: heat lability, precipitability by 0-33% ammonium sulfate and by protamine sulfate, and 8S sedimentation coefficient. High affinity binding was also detected in cytosol prepared from seminal vesicle, epididymis, and genital skin but not in non-genital skin or muscle. However, similar binding was demonstrated in the cytosol of human uterus. The steroid specificities of binding to the cytosol of male tissues of accessory reproduction and of uterus were similar in that progestational agents were more effective competitors than natural androgens. Binding specificities in cytosol prepared from genital skin were distinctly different and were similar to those of ventral prostate from the castrated rat in that dihydrotestosterone was much more potent than progestins in competition. Thus binding of R 1881 to the cytosol of prostate, epididymis, and seminal vesicle has some characteristics of binding to a progesterone receptor. When the nuclear extract from BPH was analyzed, high affinity binding was demonstrated that conformed to the specificities of binding to an androgen receptor. Here dihydrotestosterone was a more potent competitor than progestational agents. Similar patterns of binding were detected in the crude nuclear extracts from seminal vesicle, epididymis, and genital skin but not in uterus, muscle, or non-genital skin. We conclude that the androgen receptor is not demonstrable in the cytosol of prostate, epididymis, or seminal vesicle of non-castrated men but can be measured in the cytosol of genital skin and the nuclear extracts of androgen responsive tissues. Because steroid hormones exert their major influence within the nucleus of target tissues, the measurement of nuclear receptor may provide valuable insight into the regulation of growth of target tissues. PMID:73547

Identification of residues in the insulin molecule important for binding to insulin-degrading enzyme.

PubMed

Affholter, J A; Cascieri, M A; Bayne, M L; Brange, J; Casaretto, M; Roth, R A

1990-08-21

Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Synthesis and binding affinity analysis of α1-2- and α1-6-O/S-linked dimannosides for the elucidation of sulfur in glycosidic bonds using quartz crystal microbalance sensors.

PubMed

Norberg, Oscar; Wu, Bin; Thota, Niranjan; Ge, Jian-Tao; Fauquet, Germain; Saur, Ann-Kathrin; Aastrup, Teodor; Dong, Hai; Yan, Mingdi; Ramström, Olof

2017-11-27

The role of sulfur in glycosidic bonds has been evaluated using quartz crystal microbalance methodology. Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-glycosidic bonds have been developed, and the recognition properties assessed in competition binding assays with the cognate lectin concanavalin A. Mannose-presenting QCM sensors were produced using photoinitiated, nitrene-mediated immobilization methods, and the subsequent binding study was performed in an automated flow-through instrumentation, and correlated with data from isothermal titration calorimetry. The recorded K d -values corresponded well with reported binding affinities for the O-linked dimannosides with affinities for the α1-2-linked dimannosides in the lower micromolar range. The S-linked analogs showed slightly disparate effects, where the α1-6-linked analog showed weaker affinity than the O-linked dimannoside, as well as positive apparent cooperativity, whereas the α1-2-analog displayed very similar binding compared to the O-linked structure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

PubMed

Gifford, Stacey M; Liu, Weizhi; Mader, Christopher C; Halo, Tiffany L; Machida, Kazuya; Boggon, Titus J; Koleske, Anthony J

2014-07-11

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedynyshyn, J.P.

The opioid binding characteristics of the rat (PAG) and the signal transduction mechanisms of the opioid receptors were examined with in vitro radioligand binding, GTPase, adenylyl cyclase, and inositol phosphate assays. The nonselective ligand {sup 3}H-ethylketocyclazocine (EKC), the {mu} and {delta} selective ligand {sup 3}H-(D-Ala{sup 2}, D-Leu{sup 5}) enkephalin (DADLE), the {mu} selective ligand {sup 3}H-(D-Ala{sup 2}, N-methyl Phe{sup 4}, Glyol{sup 5}) enkephalin (DAGO), and the {delta} selective ligand {sup 3}H-(D-Pen{sup 2}, D-Pen{sup 5}) enkephalin (DPDPE) were separately used as tracer ligands to label opioid binding sites in rat PAG enriched P{sub 2} membrane in competition with unlabeled DADLE, DAGO,more » DPDPE, or the {kappa} selective ligand trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide, methane sulfonate, hydrate (U50, 488H). Only {mu} selective high affinity opioid binding was observed. No high affinity {delta} or {kappa} selective binding was detected. {sup 3}H-DAGO was used as a tracer ligand to label {mu} selective high affinity opioid binding sites in PAG enriched P{sub 2} membrane in competition with unlabeled {beta}-endorphin, dynorphin A (1-17), BAM-18, methionine enkephalin, dynorphin A (1-8), and leucine enkephalin. Of these endogenous opioid peptides only those with previously reported high affinity {mu} type opioid binding activity competed with {sup 3}H-DAGO for binding sites in rat PAG enriched P{sub 2} membrane with affinities similar to that of unlabeled DAGO.« less

Class B type I scavenger receptor is responsible for the high affinity cholesterol binding activity of intestinal brush border membrane vesicles

PubMed Central

Labonté, Eric D.; Howles, Philip N.; Granholm, Norman A.; Rojas, Juan C.; Davies, Joanna P.; Ioannou, Yiannis A.; Hui, David Y.

2007-01-01

Recent studies have documented the importance of Niemann Pick C1-like 1 protein (NPC1L1), a putative physiological target of the drug ezetimibe, in mediating intestinal cholesterol absorption. However, whether NPC1L1 is the high affinity cholesterol binding protein on intestinal brush border membranes is still controversial. In this study, brush border membrane vesicles (BBMV) from wild type and NPC1L1−/− mice were isolated and assayed for micellar cholesterol binding in the presence or absence of ezetimibe. Results confirmed the loss of the high affinity component of cholesterol binding when wild type BBMV preparations were incubated with antiserum against the class B type 1 scavenger receptor (SR-BI) in the reaction mixture similar to previous studies. Subsequently, second order binding of cholesterol was observed with BBMV from wild type and NPC1L1−/− mice. The inclusion of ezetimibe in these in vitro reaction assays resulted in the loss of the high affinity component of cholesterol interaction. Surprisingly, BBMVs from NPC1L1−/− mice maintained active binding of cholesterol. These results documented that SR-BI, not NPC1L1, is the major protein responsible for the initial high affinity cholesterol ligand binding process in the cholesterol absorption pathway. Additionally, ezetimibe may inhibit BBM cholesterol binding through targets such as SR-BI in addition to its inhibition of NPC1L1. PMID:17442616

Decorin is a Zn(2+) Metalloprotein

NASA Technical Reports Server (NTRS)

Yang, Vivian W.-C.; LaBrenz, Steven R.; Rosenberg, Lawrence C.; McQuillan, David; Hoeoek, Magnus

1998-01-01

Decorin is ubiquitously distributed in the extracellular matrix of mammals and a member of the proteoglycan family characterized by a core protein dominated by Leucine Rich Repeat motifs. We here demonstrate that decorin extracted from bovine tissues under denaturing conditions or produced in recombinant "native" form by cultured mammalian cells, has a high affinity for Zn(2+). Binding of Zn(2+) to decorin is demonstrated by Zn(2+) chelating chromatography and equilibrium dialyses. The Zn(2+) binding sites are localized to the N-terminal domain of the core protein that contains 4 Cys residues in the spacing reminiscent of a Zn finger. A recombinant 41 amino acid long peptide representing the N-terminal domain of decorin has full Zn(2+) binding activity and binds two Zn(2+) ions with an average K(D) of 3 x 10(exp -7) M. Biglycan, a proteoglycan that is structurally closely related to decorin contains a similar high affinity Zn(2+) binding segment, whereas the structurally more distantly related proteoglycans, epiphycan and osteoglycin, did not bind Zn(2+) with high affinity.

(/sup 3/H)Ethylketocyclazocine binding to mouse brain membranes: evidence for a kappa opioid receptor type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garzon, J.; Sanchez-Blazquez, P.; Lee, N.M.

1984-10-01

The binding of the putative kappa agonist ethylketocyclazocine (EKC) to synaptosomal membranes of mouse brain was studied. This benzomorphan was able to bind to different opioid receptors. A portion of this binding was not inhibited by the agonist naloxone, even at high concentrations (10 microM). This population of receptors, to which opioate alkaloids and opiod peptides display very low affinity, is probably the sigma receptor. Another class of binding sites was identified by the simultaneous addition of the selective agonists Sandoz FK-33824 and D-Ala2-D-Leu5-enkephalin, which blocked the access of EKC to mu and delta opioid receptors, respectively, leaving a portionmore » of naloxone-displaceable benzomorphan binding still detectable. Analysis of this remaining binding revealed a small population of receptors of high affinity, the kappa receptor. Therefore, EKC binds to the mu, delta, kappa and sigma receptors in the mouse brain, with similar affinities for the mu and kappa (0.22 and 0.15 nM). These results confirm the existence of a kappa opioid receptor type in the mouse brain.« less

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration* ♦

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine

2015-01-01

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

Binding affinity toward human prion protein of some anti-prion compounds - Assessment based on QSAR modeling, molecular docking and non-parametric ranking.

PubMed

Kovačević, Strahinja; Karadžić, Milica; Podunavac-Kuzmanović, Sanja; Jevrić, Lidija

2018-01-01

The present study is based on the quantitative structure-activity relationship (QSAR) analysis of binding affinity toward human prion protein (huPrP C ) of quinacrine, pyridine dicarbonitrile, diphenylthiazole and diphenyloxazole analogs applying different linear and non-linear chemometric regression techniques, including univariate linear regression, multiple linear regression, partial least squares regression and artificial neural networks. The QSAR analysis distinguished molecular lipophilicity as an important factor that contributes to the binding affinity. Principal component analysis was used in order to reveal similarities or dissimilarities among the studied compounds. The analysis of in silico absorption, distribution, metabolism, excretion and toxicity (ADMET) parameters was conducted. The ranking of the studied analogs on the basis of their ADMET parameters was done applying the sum of ranking differences, as a relatively new chemometric method. The main aim of the study was to reveal the most important molecular features whose changes lead to the changes in the binding affinities of the studied compounds. Another point of view on the binding affinity of the most promising analogs was established by application of molecular docking analysis. The results of the molecular docking were proven to be in agreement with the experimental outcome. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

PubMed

Werle, E; Lenz, T; Strobel, G; Weicker, H

1988-07-01

The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off

Modification of agonist binding moiety in hybrid derivative 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-1-ol/-2-amino versions: Impact on functional activity and selectivity for dopamine D2/D3 receptors

PubMed Central

Gopishetty, Bhaskar; Zhang, Suhong; Kharkar, Prashant S.; Antonio, Tamara; Reith, Maarten; Dutta, Aloke K.

2013-01-01

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D3 versus D2 receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (Ki) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D3 affinity whereas the indole derivative 13 exhibited similar high D3 affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D3 affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization. PMID:23623679

Studies on the chitin/chitosan binding properties of six cuticular proteins analogous to peritrophin 3 from Bombyx mori.

PubMed

Qu, M; Ren, Y; Liu, Y; Yang, Q

2017-08-01

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated-chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1 and BmCPAP3-D2) were cloned and expressed in Escherichia coli and purified using metal-chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3-D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3-D1 was similar to BmCPAP3-A1 and BmCPAP3-C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin-binding protein, BmCPAP3-D1, which exhibits high binding affinity to deacetylated chitin. © 2017 The Royal Entomological Society.

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein.

PubMed

Zhang, Zhen; Palzkill, Timothy

2003-11-14

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.

High affinity ligands from in vitro selection: Complex targets

PubMed Central

Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

1998-01-01

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

Allosteric Regulation of E-Cadherin Adhesion*

PubMed Central

Shashikanth, Nitesh; Petrova, Yuliya I.; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M.; Leckband, Deborah E.

2015-01-01

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120ctn dephosphorylation. PMID:26175155

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

NASA Astrophysics Data System (ADS)

Santoshi, Seneha; Naik, Pradeep K.

2014-07-01

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of -6.4, -7.2, -7.4 and -7.3 kcal/mol with αβI, αβII, αβIII and αβIV isotypes, respectively. Similarly 10 showed higher binding affinity of -6.8 kcal/mol with αβV, whereas 8 had the highest binding affinity of -7.2, -7.1 and -7.2 kcal/mol, respectively with αβVI, αβVII and αβVIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of -46.2 and -38.1 kcal/mol against αβIII (overexpression of αβIII has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.

The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

USGS Publications Warehouse

Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

2000-01-01

High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis.

PubMed

Holst, B; Hastrup, H; Raffetseder, U; Martini, L; Schwartz, T W

2001-06-08

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

PubMed

Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

2018-02-23

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis.

PubMed

Paula, Stefan; Tabet, Michael R; Keenan, Susan M; Welsh, William J; Ball, W James

2003-01-17

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy. Copyright 2003 Elsevier Science Ltd.

Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung.

PubMed Central

Walsh, D A; Salmon, M; Featherstone, R; Wharton, J; Church, M K; Polak, J M

1994-01-01

1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7534186

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

PubMed

Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

2015-09-04

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

PubMed

Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

2012-04-01

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton. Copyright © 2012 American Society for Bone and Mineral Research.

Role of tyrosine hot-spot residues at the interface of colicin E9 and immunity protein 9: a comparative free energy simulation study.

PubMed

Luitz, Manuel P; Zacharias, Martin

2013-03-01

The endonuclease activity of the bacterial colicin 9 enzyme is controlled by the specific and high-affinity binding of immunity protein 9 (Im9). Molecular dynamics simulation studies in explicit solvent were used to investigate the free energy change associated with the mutation of two hot-spot interface residues [tyrosine (Tyr): Tyr54 and Tyr55] of Im9 to Ala. In addition, the effect of several other mutations (Leu33Ala, Leu52Ala, Val34Ala, Val37Ala, Ser48Ala, and Ile53Ala) with smaller influence on binding affinity was also studied. Good qualitative agreement of calculated free energy changes and experimental data on binding affinity of the mutations was observed. The simulation studies can help to elucidate the molecular details on how the mutations influence protein-protein binding affinity. The role of solvent and conformational flexibility of the partner proteins was studied by comparing the results in the presence or absence of solvent and with or without positional restraints. Restriction of the conformational mobility of protein partners resulted in significant changes of the calculated free energies but of similar magnitude for isolated Im9 and for the complex and therefore in only modest changes of binding free energy differences. Although the overall binding free energy change was similar for the two Tyr-Ala mutations, the physical origin appeared to be different with solvation changes contributing significantly to the Tyr55Ala mutation and to a loss of direct protein-protein interactions dominating the free energy change due to the Tyr54Ala mutation. Copyright © 2012 Wiley Periodicals, Inc.

Denervation does not alter the number of neuronal bungarotoxin binding sites on autonomic neurons in the frog cardiac ganglion.

PubMed

Sargent, P B; Bryan, G K; Streichert, L C; Garrett, E N

1991-11-01

The binding of neuronal bungarotoxin (n-BuTX; also known as bungarotoxin 3.1, kappa-bungarotoxin, and toxin F) was analyzed in normal and denervated parasympathetic cardiac ganglia of the frog Rana pipiens, n-BuTX blocks both EPSPs and ACh potentials at 5-20 nM, as determined by intracellular recording techniques. Scatchard analysis on homogenates indicates that cardiac ganglia have two classes of binding sites for 125I-n-BuTX: a high-affinity site with an apparent dissociation constant (Kd,app) of 1.7 nM and a Bmax (number of binding sites) of 3.8 fmol/ganglion and a low-affinity site with a Kd,app of 12 microM and a Bmax of 14 pmol/ganglion. alpha-Bungarotoxin does not appear to interfere with the binding of 125I-n-BuTX to either site. The high-affinity binding site is likely to be the functional nicotinic ACh receptor (AChR), given the similarity between its affinity for 125I-n-BuTX and the concentration of n-BuTX required to block AChR function. Light microscopic autoradiographic analysis of 125I-n-BuTX binding to the ganglion cell surface reveals that toxin binding is concentrated at synaptic sites, which were identified using a synaptic vesicle-specific antibody. Scatchard analysis of autoradiographic data reveals that 125I-n-BuTX binding to the neuronal surface is saturable and has a Kd,app similar to that of the high-affinity binding site characterized in homogenates. Surface binding of 125I-n-BuTX is blocked by nicotine, carbachol, and d-tubocurarine (IC50 less than 20 microM), but not by atropine (IC50 greater than 10 mM). Denervation of the heart increases the ACh sensitivity of cardiac ganglion cells but has no effect upon the number of high-affinity binding sites for 125I-n-BuTX in tissue homogenates. Moreover, autoradiographic analysis indicates that denervation does not alter the number of 125I-n-BuTX binding sites on the ganglion cell surface. n-BuTX is as effective in reducing ganglion cell responses to ACh in denervated ganglia as it is in normally innervated ganglia. These results suggest that denervation alters neither the total number of nicotinic AChRs in the cardiac ganglion nor the number found on the surface of ganglion cells. These autonomic neurons thus respond differently to denervation than do skeletal myofibers. The increase in ACh sensitivity displayed by cardiac ganglion cells upon denervation cannot be explained by changes in AChR number.

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

PubMed

Gorelik, Maryna; Davidson, Alan R

2012-03-16

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

PubMed

Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

2017-10-01

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rothman, R.B.; Reid, A.; Mahboubi, A.

1991-02-01

Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and lowmore » affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.« less

Investigation of differences in the binding affinities of two analogous ligands for untagged and tagged p38 kinase using thermodynamic integration MD simulation.

PubMed

Sun, Ying-Chieh; Hsu, Wen-Chi; Hsu, Chia-Jen; Chang, Chia-Ming; Cheng, Kai-Hsiang

2015-11-01

Thermodynamic integration (TI) molecular dynamics (MD) simulations for the binding of a pair of a reference ("ref") ligand and an analogous ("analog") ligand to either tagged (with six extra residues at the N-terminus) or untagged p38 kinase proteins were carried out in order to probe how the binding affinity is influenced by the presence or absence of the peptide tag in p38 kinase. This possible effect of protein length on the binding affinity of a ligand-which is seldom addressed in the literature-is important because, even when two labs claim to have performed experiments with the same protein, they may actually have studied variants of the same protein with different lengths because they applied different protein expression conditions/procedures. Thus, if we wanted to compare ligand binding affinities measured in the two labs, it would be necessary to account for any variation in ligand binding affinity with protein length. The pair of ligand-p38 kinase complexes examined in this work (pdb codes: 3d7z and 3lhj, respectively) were ideal for investigating this effect. The experimentally determined binding energy for the ref ligand with the untagged p38 kinase was -10.9 kcal mol(-1), while that for the analog ligand with the tagged p38 kinase was -11.9 kcal mol(-1). The present TI-MD simulation of the mutation of the ref ligand into the analog ligand while the ligand is bound to the untagged p38 kinase predicted that the binding affinity of the analog ligand is 2.0 kcal mol(-1) greater than that of the ref ligand. A similar simulation also indicated that the same was true for ligand binding to the tagged protein, but in this case the binding affinity for the analog ligand is 2.5 kcal mol(-1) larger than that for the ref ligand. These results therefore suggest that the presence of the peptide tag on p38 kinase increased the difference in the binding energies of the ligands by a small amount of 0.5 kcal mol(-1). This result supports the assumption that the presence of a peptide tag has only a minor effect on ΔG values. The error bars in the computed ΔG values were then estimated via confidence interval analysis and a time autocorrelation function for the quantity dV/dλ. The estimated correlation time was ~0.5 ps and the error bar in the ΔG values estimated using nanosecond-scale simulations was ±0.3 kcal mol(-1) at a confidence level of 95%. These predicted results can be verified in future experiments and should prove useful in subsequent similar studies. Graphical Abstract Thermodynamic cycles for binding of two analogous ligands with untagged and tagged p38 kinases and associated Gibbs free energy.

Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode

PubMed Central

Cole, David K.; Sami, Malkit; Scott, Daniel R.; Rizkallah, Pierre J.; Borbulevych, Oleg Y.; Todorov, Penio T.; Moysey, Ruth K.; Jakobsen, Bent K.; Boulter, Jonathan M.; Baker, Brian M.; Yi Li

2013-01-01

Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A∗0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies. PMID:23805144

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.

PubMed

Papadakos, Grigorios A; Nastri, Horacio; Riggs, Paul; Dupureur, Cynthia M

2007-05-01

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

PubMed

Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

2002-07-01

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

USGS Publications Warehouse

Persson, Petra; Shrimpton, J. Mark; McCormick, Stephen D.; Bjornsson, Bjorn Thrandur

2000-01-01

High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol × mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol × mg protein−1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

Thermodynamic characterization of two homologous protein complexes: Associations of the semaphorin receptor plexin-B1 RhoGTPase binding domain with Rnd1 and active Rac1

PubMed Central

Hota, Prasanta K; Buck, Matthias

2009-01-01

Plexin receptors function in response to semaphorin guidance cues in a variety of developmental processes involving cell motility. Interactions with Rho, as well as Ras family small GTPases are critical events in the cell signaling mechanism. We have recently determined the structure of a cytoplasmic domain (RBD) of plexin-B1 and mapped its binding interface with several Rho-GTPases, Rac1, Rnd1, and RhoD. All three GTPases associate with a similar region of this plexin domain, but show different functional behavior in cells. To understand whether thermodynamic properties of the GTPase–RBD interaction contribute to such different behavior, we have examined the interaction at different temperatures, buffer, and pH conditions. Although the binding affinity of both Rnd1 and Rac1 with the plexin-B1 RBD is similar, the detailed thermodynamic properties of the interactions are considerably different. These data suggest that on Rac1 binding to the plexin-B1 RBD, the proteins become more rigid in the complex. By contrast, Rnd1 binding is consistent with unchanged or slightly increased flexibility in one or both proteins. Both GTPases show an appreciable reduction in affinity for the dimeric plexin-B1 RBD indicating that GTPase binding is not cooperative with dimer formation, but that a partial steric hindrance destabilizes the dimer. However, a reduced affinity binding mode to a disulphide stabilized model for the dimeric RBD is also possible. Consistent with cellular studies, the interaction thermodynamics imply that further levels of regulation involving additional binding partners and/or regions outside of the RhoGTPase binding domain are required for receptor activation. PMID:19388051

Interactions of Plakoglobin and [beta]-Catenin with Desmosomal Cadherins BASIS OF SELECTIVE EXCLUSION OF [alpha]- AND [beta]-CATENIN FROM DESMOSOMES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Hee-Jung; Gross, Julia C.; Pokutta, Sabine

2009-11-18

Plakoglobin and {beta}-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with {alpha}-catenin. Plakoglobin, but normally not {beta}-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of {beta}-catenin and {alpha}-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between {beta}-catenin and E-cadherin. Trypsin sensitivity experimentsmore » indicate that the plakoglobin arm domain by itself is more flexible than that of {beta}-catenin. Binding of plakoglobin and {beta}-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and {beta}-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, {beta}-catenin binds to desmoglein-1 more weakly than does plakoglobin. {beta}-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal {beta}-catenin 'tails' that flank the armadillo repeat region reduces the affinity for desmosomal cadherins, calorimetric measurements show no significant effects of the tails on binding to the cadherins. Using purified proteins, we show that desmosomal cadherins and {alpha}-catenin compete directly for binding to plakoglobin, consistent with the absence of {alpha}-catenin in desmosomes.« less

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

2011-11-01

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ( 1HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants Ka were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants KaI of PBZ-SA complex. Similarly, PBZ influences KaI of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of KaII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of 1HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

PubMed Central

Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

2013-01-01

When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O2-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms, providing molecular evidence for adaptive changes in O2 transport associated with acclimation to severe hypoxia. PMID:23986362

ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

PubMed

Hisabori, T; Yoshida, M; Sakurai, H

1986-09-01

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

Allosteric Regulation of E-Cadherin Adhesion.

PubMed

Shashikanth, Nitesh; Petrova, Yuliya I; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M; Leckband, Deborah E

2015-08-28

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niles, L.P.; Hashemi, F.

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax =more » 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.« less

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

PubMed

Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

2007-04-10

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

PubMed

Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

2007-05-15

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

Penicillin-binding proteins in Actinobacteria.

PubMed

Ogawara, Hiroshi

2015-04-01

Because some Actinobacteria, especially Streptomyces species, are β-lactam-producing bacteria, they have to have some self-resistant mechanism. The β-lactam biosynthetic gene clusters include genes for β-lactamases and penicillin-binding proteins (PBPs), suggesting that these are involved in self-resistance. However, direct evidence for the involvement of β-lactamases does not exist at the present time. Instead, phylogenetic analysis revealed that PBPs in Streptomyces are distinct in that Streptomyces species have much more PBPs than other Actinobacteria, and that two to three pairs of similar PBPs are present in most Streptomyces species examined. Some of these PBPs bind benzylpenicillin with very low affinity and are highly similar in their amino-acid sequences. Furthermore, other low-affinity PBPs such as SCLAV_4179 in Streptomyces clavuligerus, a β-lactam-producing Actinobacterium, may strengthen further the self-resistance against β-lactams. This review discusses the role of PBPs in resistance to benzylpenicillin in Streptomyces belonging to Actinobacteria.

Insulation and wiring specificity of BceR-like response regulators and their target promoters in Bacillus subtilis.

PubMed

Fang, Chong; Nagy-Staroń, Anna; Grafe, Martin; Heermann, Ralf; Jung, Kirsten; Gebhard, Susanne; Mascher, Thorsten

2017-04-01

BceRS and PsdRS are paralogous two-component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, P bceA or P psdA , resulting in a strong up-regulation of target gene expression and ultimately antibiotic resistance. Despite high sequence similarity between the RRs BceR and PsdR and their known binding sites, no cross-regulation has been observed between them. We therefore investigated the specificity determinants of P bceA and P psdA that ensure the insulation of these two paralogous pathways at the RR-promoter interface. In vivo and in vitro analyses demonstrate that the regulatory regions within these two promoters contain three important elements: in addition to the known (main) binding site, we identified a linker region and a secondary binding site that are crucial for functionality. Initial binding to the high-affinity, low-specificity main binding site is a prerequisite for the subsequent highly specific binding of a second RR dimer to the low-affinity secondary binding site. In addition to this hierarchical cooperative binding, discrimination requires a competition of the two RRs for their respective binding site mediated by only slight differences in binding affinities. © 2016 John Wiley & Sons Ltd.

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells.

PubMed

Fink, R D; McClay, D R

1985-01-01

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.

Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa.

PubMed

Yoshino, K; Suzuki, N

1992-06-15

We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.

An Evolutionary/Biochemical Connection Between Promoter- and Primer-Dependent Polymerases Revealed by Selective Evolution of Ligands by Exponential Enrichment (SELEX).

PubMed

Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J

2018-01-16

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promotors. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology. Copyright © 2018 American Society for Microbiology.

Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.

PubMed Central

Kelly, J J; Baird, E E; Dervan, P B

1996-01-01

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity. Images Fig. 4 PMID:8692930

Penicillin-binding proteins in Haemophilus influenzae.

PubMed Central

Makover, S D; Wright, R; Telep, E

1981-01-01

The penicillin-binding proteins (PBPs) of Haemophilus influenzae were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Eight major PBPs, ranging in molecular weights from 90,000 to 27,000, were detected. The pattern of molecular weights was different from that determined fro Escherichia coli or Pseudomonas aeruginosa. A study on the binding of several beta-lactam antibodies to the PBPs at their minimal inhibitory concentrations and at lower and higher concentrations revealed that all had highest affinity for PBP 2. Amdinocillin (mecillinam) was an exception; it had highest affinity for PBP 3. The morphological effects of several penicillins, cephalosporins, and amdinocillin on H. influenzae were similar to those reported for E. coli. Images PMID:6972731

Design and Synthesis of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile (Citalopram) Analogues as Novel Probes for the Serotonin Transporter S1 and S2 Binding Sites

PubMed Central

Banala, Ashwini K.; Zhang, Peng; Plenge, Per; Cyriac, George; Kopajtic, Theresa; Katz, Jonathan L.; Loland, Claus Juul; Newman, Amy Hauck

2013-01-01

The serotonin transporter (SERT) is the primary target for antidepressant drugs. The existence of a high affinity primary orthosteric binding site (S1) and a low affinity secondary site (S2) has been described and their relation to antidepressant pharmacology has been debated. Herein, structural modifications to the N-, 4, 5, and 4’-positions of (±)citalopram (1) are reported. All of the analogues were SERT-selective and demonstrated that steric bulk was tolerated at the SERT S1 site, including two dimeric ligands (15 and 51.) In addition, 8 analogues were identified with similar potencies to S-1 for decreasing the dissociation of [3H]S-1 from the S1 site, via allosteric modulation at S2. Both dimeric compounds had similar affinities for the SERT S1 site (Ki=19.7 and 30.2 nM, respectively), whereas only the N-substituted analogue, 51, was as effective as S-1 in allosterically modulating the binding of [3H]S-1 via S2. PMID:24237160

Three-dimensional quantitative structure-activity relationship modeling of cocaine binding by a novel human monoclonal antibody.

PubMed

Paula, Stefan; Tabet, Michael R; Farr, Carol D; Norman, Andrew B; Ball, W James

2004-01-01

Human monoclonal antibodies (mAbs) designed for immunotherapy have a high potential for avoiding the complications that may result from human immune system responses to the introduction of nonhuman mAbs into patients. This study presents a characterization of cocaine/antibody interactions that determine the binding properties of the novel human sequence mAb 2E2 using three-dimensional quantitative structure-activity relationship (3D-QSAR) methodology. We have experimentally determined the binding affinities of mAb 2E2 for cocaine and 38 cocaine analogues. The K(d) of mAb 2E2 for cocaine was 4 nM, indicating a high affinity. Also, mAb 2E2 displayed good cocaine specificity, as reflected in its 10-, 1500-, and 25000-fold lower binding affinities for the three physiologically relevant cocaine metabolites benzoylecgonine, ecgonine methyl ester, and ecgonine, respectively. 3D-QSAR models of cocaine binding were developed by comparative molecular similarity index analysis (CoMSIA). A model of high statistical quality was generated showing that cocaine binds to mAb 2E2 in a sterically restricted binding site that leaves the methyl group attached to the ring nitrogen of cocaine solvent-exposed. The methyl ester group of cocaine appears to engage in attractive van der Waals interactions with mAb 2E2, whereas the phenyl group contributes to the binding primarily via hydrophobic interactions. The model further indicated that an increase in partial positive charge near the nitrogen proton and methyl ester carbonyl group enhances binding affinity and that the ester oxygen likely forms an intermolecular hydrogen bond with mAb 2E2. Overall, the cocaine binding properties of mAb 2E2 support its clinical potential for development as a treatment of cocaine overdose and addiction.

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

PubMed Central

Jakubík, J; Janíčková, H; El-Fakahany, EE; Doležal, V

2011-01-01

BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [35S]GTPγS and [3H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M2 muscarinic acetylcholine receptor. KEY RESULTS Agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [3H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from Gi/o G-proteins but only its dissociation from Gs/olf G-proteins. CONCLUSIONS AND IMPLICATIONS These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of Gi/o versus Gs/olf G-proteins that are not identified by conventional GTPγS binding. PMID:20958290

Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

PubMed

Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

2011-03-01

Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Concentration-Dependent Multiple Binding Sites on Saliva-Treated Hydroxyapatite for Streptococcus sanguis

PubMed Central

Gibbons, R. J.; Moreno, E. C.; Etherden, I.

1983-01-01

The influence of bacterial cell concentration on estimates of the number of binding sites and the affinity for the adsorption of a strain of Streptococcus sanguis to saliva-treated hydroxyapatite was determined, and the possible presence of multiple binding sites for this organism was tested. The range of concentrations of available bacteria varied from 4.7 × 106 to 5,960 × 106 cells per ml. The numbers of adsorbed bacteria increased over the entire range tested, but a suggestion of a break in an otherwise smooth adsorption isotherm was evident. Values for the number of binding sites and the affinity varied considerably depending upon the range of available bacterial concentrations used to estimate them; high correlation coefficients were obtained in all cases. The use of low bacterial cell concentrations yielded lower values for the number of sites and much higher values for the affinity constant than did the use of high bacterial cell concentrations. When data covering the entire range of bacterial concentrations were employed, values for the number of sites and the affinity were similar to those obtained by using only high bacterial cell concentrations. The simplest explanation for these results is that there are multiple binding sites for S. sanguis on saliva-treated hydroxyapatite surfaces. When present in low concentration, the streptococci evidently attach to more specific high-affinity sites which become saturated when higher bacterial concentrations are employed. The possibility of multiple binding sites was substantiated by comparing estimates of the adsorption parameters from a computer-simulated isotherm with those derived from the experimentally generated isotherm. A mathematical model describing bacterial adsorption to binary binding sites was further evidence for the existence of at least two classes of binding sites for S. sanguis. Far fewer streptococci adsorbed to experimental pellicles prepared from saliva depleted of bacterial aggregating activity when low numbers of streptococci were used, but the magnitude of this difference was considerably less when high streptococcal concentrations were employed. This suggests an association between salivary components which possess bacterial-aggregating activity and bacterial adsorption to high-affinity specific binding sites on saliva-treated hydroxyapatite surfaces. PMID:6822416

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

PubMed

Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

2002-02-01

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets.

PubMed

Nelson, Christopher S; Fuller, Chris K; Fordyce, Polly M; Greninger, Alexander L; Li, Hao; DeRisi, Joseph L

2013-07-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein's DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2's-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved.

Microfluidic affinity and ChIP-seq analyses converge on a conserved FOXP2-binding motif in chimp and human, which enables the detection of evolutionarily novel targets

PubMed Central

Nelson, Christopher S.; Fuller, Chris K.; Fordyce, Polly M.; Greninger, Alexander L.; Li, Hao; DeRisi, Joseph L.

2013-01-01

The transcription factor forkhead box P2 (FOXP2) is believed to be important in the evolution of human speech. A mutation in its DNA-binding domain causes severe speech impairment. Humans have acquired two coding changes relative to the conserved mammalian sequence. Despite intense interest in FOXP2, it has remained an open question whether the human protein’s DNA-binding specificity and chromatin localization are conserved. Previous in vitro and ChIP-chip studies have provided conflicting consensus sequences for the FOXP2-binding site. Using MITOMI 2.0 microfluidic affinity assays, we describe the binding site of FOXP2 and its affinity profile in base-specific detail for all substitutions of the strongest binding site. We find that human and chimp FOXP2 have similar binding sites that are distinct from previously suggested consensus binding sites. Additionally, through analysis of FOXP2 ChIP-seq data from cultured neurons, we find strong overrepresentation of a motif that matches our in vitro results and identifies a set of genes with FOXP2 binding sites. The FOXP2-binding sites tend to be conserved, yet we identified 38 instances of evolutionarily novel sites in humans. Combined, these data present a comprehensive portrait of FOXP2’s-binding properties and imply that although its sequence specificity has been conserved, some of its genomic binding sites are newly evolved. PMID:23625967

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

PubMed Central

Monarez, Roberto R.; Macdonald, Clinton C.; Dass, Brinda

2006-01-01

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, τCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between τCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of τCstF-64 and CstF-64 have different affinities for RNA elements. We quantified Kd values of CstF-64 and τCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than τCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal α-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is important in RNA sequence recognition. This supports the hypothesis that τCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements. PMID:17029590

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies.

PubMed

Bazzoli, Andrea; Vance, David J; Rudolph, Michael J; Rong, Yinghui; Angalakurthi, Siva Krishna; Toth, Ronald T; Middaugh, C Russell; Volkin, David B; Weis, David D; Karanicolas, John; Mantis, Nicholas J

2017-11-01

In this report we investigated, within a group of closely related single domain camelid antibodies (V H Hs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like V H Hs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1 R29G mutant was largely devoid of toxin-neutralizing activity (TNA). However, the TNA of the V1C7 G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function. © 2017 Wiley Periodicals, Inc.

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

PubMed

Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

1998-08-21

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

Free energy calculations of glycosaminoglycan-protein interactions.

PubMed

Gandhi, Neha S; Mancera, Ricardo L

2009-10-01

Glycosaminoglycans (GAGs) are complex highly charged linear polysaccharides that have a variety of roles in biological processes. We report the first use of molecular dynamics (MD) free energy calculations using the MM/PBSA method to investigate the binding of GAGs to protein molecules, namely the platelet endothelial cell adhesion molecule 1 (PECAM-1) and annexin A2. Calculations of the free energy of the binding of heparin fragments of different sizes reveal the existence of a region of low GAG-binding affinity in domains 5-6 of PECAM-1 and a region of high affinity in domains 2-3, consistent with experimental data and ligand-protein docking studies. A conformational hinge movement between domains 2 and 3 was observed, which allows the binding of heparin fragments of increasing size (pentasaccharides to octasaccharides) with an increasingly higher binding affinity. Similar simulations of the binding of a heparin fragment to annexin A2 reveal the optimization of electrostatic and hydrogen bonding interactions with the protein and protein-bound calcium ions. In general, these free energy calculations reveal that the binding of heparin to protein surfaces is dominated by strong electrostatic interactions for longer fragments, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large unfavorable desolvation penalty due to the high charge density of these molecules.

Importance of polar solvation and configurational entropy for design of antiretroviral drugs targeting HIV-1 protease.

PubMed

Kar, Parimal; Lipowsky, Reinhard; Knecht, Volker

2013-05-16

Both KNI-10033 and KNI-10075 are high affinity preclinical HIV-1 protease (PR) inhibitors with affinities in the picomolar range. In this work, the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method has been used to investigate the potency of these two HIV-1 PR inhibitors against the wild-type and mutated proteases assuming that potency correlates with the affinity of the drugs for the target protein. The decomposition of the binding free energy reveals the origin of binding affinities or mutation-induced affinity changes. Our calculations indicate that the mutation I50V causes drug resistance against both inhibitors. On the other hand, we predict that the mutant I84V causes drug resistance against KNI-10075 while KNI-10033 is more potent against the I84V mutant compared to wild-type protease. Drug resistance arises mainly from unfavorable shifts in van der Waals interactions and configurational entropy. The latter indicates that neglecting changes in configurational entropy in the computation of relative binding affinities as often done is not appropriate in general. For the bound complex PR(I50V)-KNI-10075, an increased polar solvation free energy also contributes to the drug resistance. The importance of polar solvation free energies is revealed when interactions governing the binding of KNI-10033 or KNI-10075 to the wild-type protease are compared to the inhibitors darunavir or GRL-06579A. Although the contributions from intermolecular electrostatic and van der Waals interactions as well as the nonpolar component of the solvation free energy are more favorable for PR-KNI-10033 or PR-KNI-10075 compared to PR-DRV or PR-GRL-06579A, both KNI-10033 and KNI-10075 show a similar affinity as darunavir and a lower binding affinity relative to GRL-06579A. This is because of the polar solvation free energy which is less unfavorable for darunavir or GRL-06579A relative to KNI-10033 or KNI-10075. The importance of the polar solvation as revealed here highlights that structural inspection alone is not sufficient for identifying the key contributions to binding affinities and affinity changes for the design of drugs but that solvation effects must be taken into account. A detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs.

Improving binding mode and binding affinity predictions of docking by ligand-based search of protein conformations: evaluation in D3R grand challenge 2015

NASA Astrophysics Data System (ADS)

Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

2017-08-01

The growing number of protein-ligand complex structures, particularly the structures of proteins co-bound with different ligands, in the Protein Data Bank helps us tackle two major challenges in molecular docking studies: the protein flexibility and the scoring function. Here, we introduced a systematic strategy by using the information embedded in the known protein-ligand complex structures to improve both binding mode and binding affinity predictions. Specifically, a ligand similarity calculation method was employed to search a receptor structure with a bound ligand sharing high similarity with the query ligand for the docking use. The strategy was applied to the two datasets (HSP90 and MAP4K4) in recent D3R Grand Challenge 2015. In addition, for the HSP90 dataset, a system-specific scoring function (ITScore2_hsp90) was generated by recalibrating our statistical potential-based scoring function (ITScore2) using the known protein-ligand complex structures and the statistical mechanics-based iterative method. For the HSP90 dataset, better performances were achieved for both binding mode and binding affinity predictions comparing with the original ITScore2 and with ensemble docking. For the MAP4K4 dataset, although there were only eight known protein-ligand complex structures, our docking strategy achieved a comparable performance with ensemble docking. Our method for receptor conformational selection and iterative method for the development of system-specific statistical potential-based scoring functions can be easily applied to other protein targets that have a number of protein-ligand complex structures available to improve predictions on binding.

Neuromedin B receptor in esophagus: evidence for subtypes of bombesin receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Von Schrenck, T.; Heinz-Erian, P.; Moran, T.

1989-04-01

To identify receptors for bombesin-related peptides in the rat esophagus, we measured binding of 125I-Bolton-Hunter neuromedin B (125I-BH-neuromedin B) and 125I-(Tyr4)bombesin to tissue sections from the rat esophagus and compared the results with those for rat pancreas. Esophagus bound both tracers, whereas pancreas bound only 125I-(Tyr4)bombesin. In each tissue binding was saturable, dependent on pH, on time, and on temperature, reversible, and specific. Autoradiography demonstrated binding of both tracers only to the muscularis mucosae of the esophagus and binding of 125I-(Tyr4)bombesin diffusely over pancreatic acini. In the esophagus, the relative potencies for inhibition of binding of both tracers were asmore » follows: neuromedin B greater than bombesin greater than GRP = neuromedin C; similar relative potencies were found for causing contraction of muscle strips from whole esophagus and from the isolated muscularis mucosae. In pancreas tissue sections and dispersed acini, the relative potencies for inhibition of binding of 125I-(Tyr4)bombesin were as follows: bombesin greater than GRP = neuromedin C much greater than neuromedin B. Similar relative potencies were found for stimulation of enzyme secretion from dispersed pancreatic acini. Computer analysis in both tissues demonstrated only a single binding site. The present study demonstrates that rat esophagus muscle possesses specific receptors for bombesin-related peptides. Furthermore, this study shows that the esophageal bombesin receptors represent a previously unidentified class of bombesin receptors in that they have a higher affinity for neuromedin B than for bombesin. In contrast, the pancreatic bombesin receptors have, like all other bombesin receptors described to date, a high affinity for bombesin, but low affinity for neuromedin B.« less

Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crankshaw, D.; Gaspar, V.; Pliska, V.

1990-01-01

The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The bindingmore » parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.« less

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization ofmore » the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.« less

Intrinsic Thermodynamics and Structures of 2,4- and 3,4-Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases.

PubMed

Zubrienė, Asta; Smirnov, Alexey; Dudutienė, Virginija; Timm, David D; Matulienė, Jurgita; Michailovienė, Vilma; Zakšauskas, Audrius; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

2017-01-20

The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol -1 , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pK a values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

PubMed

Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

2008-08-22

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Sodium ion modulates D2 receptor characteristics of dopamine agonist and antagonist binding sites in striatum and retina

PubMed Central

Makman, Maynard H.; Dvorkin, B.; Klein, Patrice N.

1982-01-01

Sodium ion (Na+) influences binding of both dopamine agonists and antagonists to D2 receptors in striatum and retina. Also, Na+ markedly potentiates the loss of high-affinity agonist binding due to the GTP analogue p[NH]ppG. 2-Amino-6, 7-dihydroxy-1,2,3,4-tetrahydro[5,8-3H]naphthalene ([3H]ADTN) binds exclusively to an agonist conformation of D2 receptor in both striatum and retina, distinct from the antagonist conformation labeled by [3H]spiroperidol or [3H]domperidone in striatum or by [3H]spiroperidol in retina. Na+ is not required for interaction of [3H]ADTN or antagonist radioligand sites with the selective D2 agonist LY-141865, the D2 antagonist domperidone, or nonselective dopamine agonists or antagonists; however, Na+ is necessary for high affinity interaction of those radioligand sites with the D2 antagonists molindone and metoclopramide. With Na+ present, striatal sites for [3H]ADTN, [3H]spiroperidol, and [3H]domperidone have similar affinities for antagonists but only [3H]ADTN sites have high affinity for agonists. Na+ further decreases the low affinity of dopamine agonists for [3H]spiroperidol binding sites. Also, Na+ enhances [3H]spiroperidol and decreases [3H]ADTN binding. Na+ alone causes bound [3H]ADTN to dissociate from at least 30% of striatal and 50% of retinal sites, and with Na+ present [3H]ADTN rapidly dissociates from the remaining sites upon addition of p[NH]ppG. It is proposed that D2 receptors in striatum and retina exist in distinct but interconvertible conformational states, with different properties depending on the presence or absence of Na+ and of guanine nucleotide. PMID:6213964

Modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays

PubMed Central

Daniels, V; Wood, M; Leclercq, K; Kaminski, R M; Gillard, M

2013-01-01

Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies. PMID:23530581

Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Jing; Akwabi-Ameyaw, Adwoa; Britton, Jonathan E.

2009-06-24

A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ER{alpha} and ER{beta} binding affinity ranging from micromolar to low nanomolar.

Binding of Disordered Peptides to Kelch: Insights from Enhanced Sampling Simulations.

PubMed

Do, Trang Nhu; Choy, Wing-Yiu; Karttunen, Mikko

2016-01-12

Keap1 protein plays an essential role in regulating cellular oxidative stress response and is a crucial binding hub for multiple proteins, several of which are intrinsically disordered proteins (IDP). Among Kelch's IDP binding partners, NRF2 and PTMA are the two most interesting cases. They share a highly similar binding motif; however, NRF2 binds to Kelch with a binding affinity of approximately 100-fold higher than that of PTMA. In this study, we perform an exhaustive sampling composed of 6 μs well-tempered metadynamics and 2 μs unbiased molecular dynamics (MD) simulations aiming at characterizing the binding mechanisms and structural properties of these two peptides. Our results agree with previous experimental observations that PTMA is remarkably more disordered than NRF2 in both the free and bound states. This explains PTMA's lower binding affinity. Our extensive sampling also provides valuable insights into the vast conformational ensembles of both NRF2 and PTMA, supports the hypothesis of coupled folding-binding, and confirms the essential role of linear motifs in IDP binding.

Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gundlach, A.L.; Largent, B.L.; Snyder, S.H.

1986-06-01

(+)3H-3-PPP ((+)3H-3-(3-Hydroxyphenyl)-N-(1-propyl)-piperidine) binds with high affinity to brain membranes with a pharmacological profile consistent with that of sigma receptors. The distribution of (+)3H-3-PPP binding sites in brain and spinal cord of both guinea pig and rat has been determined by in vitro autoradiography with binding densities quantitated by computer-assisted densitometry. (+)3H-3-PPP binding to slide-mounted brain sections is saturable and displays high affinity and a pharmacological specificity very similar to sites labeled in homogenates. (+)3H-3-PPP binding sites are heterogeneously distributed. Highest concentrations of binding sites occur in spinal cord, particularly the ventral horn and dorsal root ganglia; the pons-medulla, associated withmore » the cranial nerve and pontine nuclei and throughout the brain stem reticular formation; the cerebellum, over the Purkinje cell layer; the midbrain, particularly the central gray and red nucleus; and hippocampus, over the pyramidal cell layer. Lowest levels are seen in the basal ganglia and parts of the thalamus, while all other areas, including hypothalamus and cerebral cortex, exhibit moderate grain densities. Quinolinic acid-induced lesions of the hippocampus indicate that (+)3H-3-PPP labels hippocampal pyramidal cells and granule cells in the dentate gyrus. Intrastriatal injection of ibotenic acid dramatically reduces (+)3H-3-PPP binding in this area, while injection of 6-hydroxydopamine produces a relatively slight decrease. The distribution of (+)3H-3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransmitter or neuropeptide, including dopamine. However, there is a notable similarity between the distribution of (+)3H-3-PPP sites and high-affinity binding sites for psychotomimetic opioids, such as the benzomorphan (+)SKF 10,047.« less

Proteome-wide inference of human endophilin 1-binding peptides.

PubMed

Wu, Gang; Zhang, Zeng-Li; Fu, Chun-Jiang; Lv, Feng-Lin; Tian, Fei-Fei

2012-10-01

Human endophilin 1 (hEndo1) is a multifunctional protein that was found to bind a wide spectrum of prolinerich endocytic proteins through its Src homology 3 (SH3) domain. In order to elucidate the unknown biological functions of hEndo1, it is essential to find out the cytoplasmic components that hEndo1 recognizes and binds. However, it is too time-consuming and expensive to synthesize all peptide candidates found in the human proteome and to perform hEndo1 SH3-peptide affinity assay to identify the hEndo1-binding partners. In the present work, we describe a structure/ sequence-hybrid approach to perform proteome-wide inference of human hEndo1-binding peptides using the information gained from both the primary sequence of affinity-known peptides and the interaction profile involved in hEndo1 SH3-peptide complex three-dimensional structures. Modeling results show that (i) different residue positions contribute distinctly to peptide affinity and specificity; P-1, P2 and P4 are most important, P1 and P3 are also effective, and P-3, P-2, P0, P5 and P6 are relatively insignificant, (ii) the consensus core PXXP motif is necessary but not sufficient for determining high affinity of peptides, and some other positions must be also essential in the hEndo1 SH3-peptide binding, and (iii) the alternating arrangement of polar and nonpolar amino acids along peptide sequence is critical for the high specificity of peptide recognition by hEndo1 SH3 domain. In addition, we also find that the residue type at a specific position of hEndo1-binding peptides is not stringently invariable; amino acids that possess similar polarity could replace each other without substantial influence on peptide affinity. In this way, hEndo1 presents a broad specificity in the peptide ligands that it binds.

Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.

PubMed

Aidoudi, Sallouha; Bikfalvi, Andreas

2010-11-01

Platelet factor-4 (PF4), a platelet-derived chemokine, has two important functions in the vasculature. It has a pro-atherogenic role while also having anti-angiogenic effects. The activity of platelet factor-4 (PF4), unlike other chemokines that bind to specific receptors, depends on its unusually high affinity for proteoglycans and other negatively charged molecules. High affinity for heparan sulfates was thought to be central to all of PF4's biological functions. However, other mechanisms have been described such as direct growth factor binding, activation of the CXCR3B chemokine receptor isoform that is present in some vascular cells or binding to lipoprotein-related protein-1 (LRP1). Furthermore, PF4 also binds to integrins with affinities similar to matrix molecules. These interactions may explain the effects of PF4 in healthy and pathological tissues. However, the mechanisms involved in PF4's activity are complex and may depend on a given tissue or localisation. Overall, while much is already known about PF4, its specific role in atherosclerosis and angiogenesis remains still to be clarified.

Characterization of calmodulin binding domains in TRPV2 and TRPV5 C-tails.

PubMed

Holakovska, Blanka; Grycova, Lenka; Bily, Jan; Teisinger, Jan

2011-02-01

The transient receptor potential channels TRPV2 and TRPV5 belong to the vanilloid TRP subfamily. TRPV2 is highly similar to TRPV1 and shares many common properties with it. TRPV5 (and also its homolog TRPV6) is a rather distinct member of the TRPV subfamily. It is distant for being strictly Ca(2+)-selective and features quite different properties from the rest of the TRPV subfamily. It is known that TRP channels are regulated by calmodulin in a calcium-dependent manner. In our study we identified a calmodulin binding site on the C-termini of TRPV2 (654-683) and TRPV5 (587-616) corresponding to the consensus CaM binding motif 1-5-10. The R679 and K681 single mutants of TRPV2 caused a 50% decrease in binding affinity and a double mutation of K661/K664 of the same peptide lowered the binding affinity by up to 75%. A double mutation of R606/K607 and triple mutation of R594/R606/R610 in TRPV5 C-terminal peptide resulted in the total loss of binding affinity to calmodulin. These results demonstrate that the TRPV2 C-tail and TRPV5 C-tail contain calmodulin binding sites and that the basic residues are strongly involved in TRP channel binding to calmodulin.

Architecture and dynamics of overlapped RNA regulatory networks.

PubMed

Lapointe, Christopher P; Preston, Melanie A; Wilinski, Daniel; Saunders, Harriet A J; Campbell, Zachary T; Wickens, Marvin

2017-11-01

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities-Puf3p, Puf4p, and Puf5p of S. cerevisiae Using RNA Tagging, we identified a "super-network" comprised of four subnetworks: Puf3p, Puf4p, and Puf5p subnetworks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, is determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: The remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution. © 2017 Lapointe et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Lessons learned from participating in D3R 2016 Grand Challenge 2: compounds targeting the farnesoid X receptor

NASA Astrophysics Data System (ADS)

Duan, Rui; Xu, Xianjin; Zou, Xiaoqin

2018-01-01

D3R 2016 Grand Challenge 2 focused on predictions of binding modes and affinities for 102 compounds against the farnesoid X receptor (FXR). In this challenge, two distinct methods, a docking-based method and a template-based method, were employed by our team for the binding mode prediction. For the new template-based method, 3D ligand similarities were calculated for each query compound against the ligands in the co-crystal structures of FXR available in Protein Data Bank. The binding mode was predicted based on the co-crystal protein structure containing the ligand with the best ligand similarity score against the query compound. For the FXR dataset, the template-based method achieved a better performance than the docking-based method on the binding mode prediction. For the binding affinity prediction, an in-house knowledge-based scoring function ITScore2 and MM/PBSA approach were employed. Good performance was achieved for MM/PBSA, whereas the performance of ITScore2 was sensitive to ligand composition, e.g. the percentage of carbon atoms in the compounds. The sensitivity to ligand composition could be a clue for the further improvement of our knowledge-based scoring function.

Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

NASA Astrophysics Data System (ADS)

Keefe, Andrew J.; Jiang, Shaoyi

2012-01-01

Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F.

2008-08-19

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to themore » lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.« less

Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia

D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoinedmore » by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.« less

Absence of C-type natriuretic peptide receptors in hamster glomeruli.

PubMed

Luk, J K; Wong, E F; Wong, N L

1994-01-01

The distribution of atrial natriuretic peptide receptor B (ANPR-B) varies between tissues and species. The aim of this study is to determine whether ANPR-B is present in the hamster glomeruli. In vitro C-type natriuretic peptide (CNP)- and atrial natriuretic factor (ANF)-stimulated cGMP accumulation studies were performed in hamster glomeruli. Elevated cGMP accumulations were observed upon ANF addition. No cGMP response was seen with CNP. Competitive receptor-binding experiments were performed with 125I-CNP and 125I-ANF against their respective cold peptides in hamster glomeruli. Although no CNP binding was detected, positive ANF binding was found and two types of ANF receptor were demonstrated. The affinity (Kdl) and maximum binding capacity (Bmaxl) of the high-affinity ANF receptor were 0.014 +/- 0.001 nM and 60.4 +/- 10.2 fmol/mg protein, respectively. Those of the low-affinity receptor (Kd2 and Bmax2) were 45.7 +/- 6.2 nM and 28.3 +/- 6.3 pmol/mg protein, respectively. Similarly, saturation binding experiments also failed to show any CNP receptor binding in hamster glomeruli. This finding suggests that ANPR-B is not present in hamster glomeruli and CNP is not a direct physiological regulator of hamster renal function.

Dual inhibition of human type 4 phosphodiesterase isostates by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3- methyl-1-pyrrolidinecarboxylate.

PubMed

Tian, G; Rocque, W J; Wiseman, J S; Thompson, I Z; Holmes, W D; Domanico, P L; Stafford, J A; Feldman, P L; Luther, M A

1998-05-12

Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) exists in both a low- and a high-affinity state that bind (R)-rolipram with Kd's of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., Wiseman, J. S., Holmes, W. D., Thompson, I. Z., Willard, D. H., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., and Luther, M. A. (1997) Biochemistry 36, 14250-14261]. Since the tissue distribution of the two isostates may be significantly different, development of inhibitors that effectively inhibit both forms may be advantageous pharmacologically. In this study, enzyme inhibition and binding of HSPDE4B2B by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidin ecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PDE 4), were investigated. Binding experiments demonstrated high-affinity binding of 1 to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of PDE activity showed only a single transition with an observed Ki similar to the apparent Kd determined by the binding experiments. Deletional mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram with low affinity, were shown to interact with 1 with high affinity, indistinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition of HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitions exemplified in the interaction of (R)-rolipram with HSPDE4B2B indicated that the two isostates were nonexchangeable. Phosphorylation at serines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In contrast to (R)-rolipram, where only one of the two isostates of PDE 4 binds with high affinity, 1 is a potent, dual inhibitor of both of the isostates of PDE 4. Kinetic and thermodynamic models describing the interactions between the nonexchangeable isostates of PDE 4 and its ligands are discussed.

2-Substituted 3β-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

PubMed

Hong, Weimin C; Kopajtic, Theresa A; Xu, Lifen; Lomenzo, Stacey A; Jean, Bernandie; Madura, Jeffry D; Surratt, Christopher K; Trudell, Mark L; Katz, Jonathan L

2016-03-01

Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3β-aryltropanes with 2β-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2β-Ph2COCH2-3β-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2β compounds increased locomotion, only the 2β-(4-ClPh)PhCOCH2-3β-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2β- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine. U.S. Government work not protected by U.S. copyright.

Novel Carbonyl Analogues of Tamoxifen: Design, Synthesis, and Biological Evaluation

NASA Astrophysics Data System (ADS)

Kasiotis, Konstantinos M.; Lambrinidis, George; Fokialakis, Nikolas; Tzanetou, Evangelia N.; Mikros, Emmanuel; Haroutounian, Serkos A.

2017-09-01

Aim of this work was to provide tamoxifen analogues with enhanced estrogen receptor binding affinity. Hence, several derivatives were prepared using an efficient triarylethylenes synthetic protocol. The novel compounds bioactivity was evaluated through the determination of their receptor binding affinity and their agonist/antagonist activity against breast cancer tissue using a MCF-7 cell-based assay. Phenyl esters 6a,b and 8a,b exhibited binding affinity to both ERα and ERβ higher than 4-hydroxytamoxifen while compounds 13 and 14 have shown cellular antiestrogenic activity similar to 4-hydroxytamoxifen and the known estrogen receptor inhibitor ICI182,780. Theoretical calculations and molecular modelling were applied to investigate, support and explain the biological profile of the new compounds. The relevant data indicated an agreement between calculations and demonstrated biological activity allowing to extract useful structure-activity relationships. Results herein underline that modifications of tamoxifen structure still provide molecules with substantial activity, as portrayed in the inhibition of MCF-7 cells proliferation.

Protein unfolding as a switch from self-recognition to high-affinity client binding

PubMed Central

Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

2016-01-01

Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

Insight into resistance mechanism of anaplastic lymphoma kinase to alectinib and JH-VIII-157-02 caused by G1202R solvent front mutation.

PubMed

Wang, Han; Wang, Yao; Guo, Wentao; Du, Bin; Huang, Xiaobing; Wu, Riping; Yang, Baoyu; Lin, Xiaoyan; Wu, Yilan

2018-01-01

Mutated anaplastic lymphoma kinase (ALK) drives the development of advanced non-small cell lung cancer (NSCLC). Most reported small-molecule inhibitors targeting the ALK domain do not display good inhibition of the G1202R solvent front mutation. The solvent front mutation was assumed to hinder drug binding. However, a different fact could be uncovered by the simulations reported in this study through a structural analog of alectinib (JH-VIII-157-02), which demonstrated potent effects against the G1202R mutation. Molecular docking, conventional molecular dynamics (MD) simulations, free energy calculations, and umbrella sampling (US) simulations were carried out to make clear the principles of the binding preferences of alectinib and JH-VIII-157-02 toward ALK WT and the ALK G1202R (ALK G1202R ) mutation. JH-VIII-157-02 has similar binding affinities to both ALK WT and ALK G1202R whereas it has has a much lower binding affinity for alectinib to ALK G1202R . Analysis of individual energy terms indicate the major variation involves the van der Waals and entropy terms. Structural analysis reveals that the conformational change of the ATP-binding glycine-rich loop was primarily responsible for the alectinib resistance, not JH-VIII-157-02. In addition, US simulations prove JH-VIII-157-02 has similar dissociative processes from both ALK WT and ALK G1202R , while alectinib is more easily dissociated from ALK G1202R than from ALK WT , thus indicating lesser residence time. Both the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation in ALK resistance.

Insight into resistance mechanism of anaplastic lymphoma kinase to alectinib and JH-VIII-157-02 caused by G1202R solvent front mutation

PubMed Central

Wang, Han; Wang, Yao; Guo, Wentao; Du, Bin; Huang, Xiaobing; Wu, Riping; Yang, Baoyu; Lin, Xiaoyan; Wu, Yilan

2018-01-01

Background Mutated anaplastic lymphoma kinase (ALK) drives the development of advanced non-small cell lung cancer (NSCLC). Most reported small-molecule inhibitors targeting the ALK domain do not display good inhibition of the G1202R solvent front mutation. The solvent front mutation was assumed to hinder drug binding. However, a different fact could be uncovered by the simulations reported in this study through a structural analog of alectinib (JH-VIII-157-02), which demonstrated potent effects against the G1202R mutation. Methods Molecular docking, conventional molecular dynamics (MD) simulations, free energy calculations, and umbrella sampling (US) simulations were carried out to make clear the principles of the binding preferences of alectinib and JH-VIII-157-02 toward ALKWT and the ALK G1202R (ALKG1202R) mutation. Results JH-VIII-157-02 has similar binding affinities to both ALKWT and ALKG1202R whereas it has has a much lower binding affinity for alectinib to ALKG1202R. Analysis of individual energy terms indicate the major variation involves the van der Waals and entropy terms. Structural analysis reveals that the conformational change of the ATP-binding glycine-rich loop was primarily responsible for the alectinib resistance, not JH-VIII-157-02. In addition, US simulations prove JH-VIII-157-02 has similar dissociative processes from both ALKWT and ALKG1202R, while alectinib is more easily dissociated from ALKG1202R than from ALKWT, thus indicating lesser residence time. Conclusion Both the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation in ALK resistance. PMID:29785088

Binding modes of environmental endocrine disruptors to human serum albumin: insights from STD-NMR, ITC, spectroscopic and molecular docking studies.

PubMed

Yang, Hongqin; Huang, Yanmei; Liu, Jiuyang; Tang, Peixiao; Sun, Qiaomei; Xiong, Xinnuo; Tang, Bin; He, Jiawei; Li, Hui

2017-09-11

Given that bisphenols have an endocrine-disrupting effect on human bodies, thoroughly exposing their potential effects at the molecular level is important. Saturation transfer difference (STD) NMR-based binding studies were performed to investigate the binding potential of two bisphenol representatives, namely, bisphenol B (BPB) and bisphenol E (BPE), toward human serum albumin (HSA). The relative STD (%) suggested that BPB and BPE show similar binding modes and orientations, in which the phenolic rings were spatially close to HSA binding site. ITC analysis results showed that BPB and BPE were bound to HSA with moderately strong binding affinity through electrostatic interactions and hydrogen bonds. The order of binding affinity of HSA for two test bisphenols is as follows: BPE > BPB. The results of fluorescence competitive experiments using 5-dimethylaminonaphthalene-1-sulfonamide and dansylsarcosine as competitors, combined with molecular docking indicated that both bisphenols are prone to attach to the binding site II in HSA. Spectroscopic results (FT-IR, CD, synchronous and 3D fluorescence spectra) showed that BPB/BPE induces different degrees of microenvironmental and conformational changes to HSA.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding sitemore » has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.« less

The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

PubMed

Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

2013-11-01

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens. Copyright © 2013 Elsevier Ltd. All rights reserved.

A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.

PubMed

Gervais, A; Lane, L K; Anner, B M; Lindenmayer, G E; Schwartz, A

1977-01-01

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.

Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS | Center for Cancer Research

Cancer.gov

A Better Fit. An improved anticoagulant drug called RUC-2 (ball and stick structure) fits snugly into its binding pocket on integrin (blue), a protein found on the surface of platelets. RUC-2 binds both subunits of integrin, inhibiting the excessive blood coagulation that can lead to strokes and heart attacks. Unlike similar drugs that alter integrin's structure when they bind

An HPLC method associated with a thermodynamic analysis to compare the binding of TRAIL and its nanovectorized form to death receptors DR4 and DR5 and their relationship to cytotoxicity.

PubMed

Guillaume, Yves Claude; Lethier, Lydie; André, Claire

2016-11-15

TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1°C-50°C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to -0.91kJ/molK and -0.28kJ/molK and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to -1.54kJ/molK and -1.05kJ/molK. By the use of differential scanning calorimetry (DSC), a phase transition (∼12°C for DR5, ∼4°C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy ΔH for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive ΔH values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative ΔH values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand - receptor interface. Above 30°C, their rank-ordered affinities were thus strongly different in the sequence: TRAIL DR4

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

PubMed

Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

2017-07-05

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Performance of HADDOCK and a simple contact-based protein-ligand binding affinity predictor in the D3R Grand Challenge 2

NASA Astrophysics Data System (ADS)

Kurkcuoglu, Zeynep; Koukos, Panagiotis I.; Citro, Nevia; Trellet, Mikael E.; Rodrigues, J. P. G. L. M.; Moreira, Irina S.; Roel-Touris, Jorge; Melquiond, Adrien S. J.; Geng, Cunliang; Schaarschmidt, Jörg; Xue, Li C.; Vangone, Anna; Bonvin, A. M. J. J.

2018-01-01

We present the performance of HADDOCK, our information-driven docking software, in the second edition of the D3R Grand Challenge. In this blind experiment, participants were requested to predict the structures and binding affinities of complexes between the Farnesoid X nuclear receptor and 102 different ligands. The models obtained in Stage1 with HADDOCK and ligand-specific protocol show an average ligand RMSD of 5.1 Å from the crystal structure. Only 6/35 targets were within 2.5 Å RMSD from the reference, which prompted us to investigate the limiting factors and revise our protocol for Stage2. The choice of the receptor conformation appeared to have the strongest influence on the results. Our Stage2 models were of higher quality (13 out of 35 were within 2.5 Å), with an average RMSD of 4.1 Å. The docking protocol was applied to all 102 ligands to generate poses for binding affinity prediction. We developed a modified version of our contact-based binding affinity predictor PRODIGY, using the number of interatomic contacts classified by their type and the intermolecular electrostatic energy. This simple structure-based binding affinity predictor shows a Kendall's Tau correlation of 0.37 in ranking the ligands (7th best out of 77 methods, 5th/25 groups). Those results were obtained from the average prediction over the top10 poses, irrespective of their similarity/correctness, underscoring the robustness of our simple predictor. This results in an enrichment factor of 2.5 compared to a random predictor for ranking ligands within the top 25%, making it a promising approach to identify lead compounds in virtual screening.

Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voleti, Rashmi; Tomchick, Diana R.; Südhof, Thomas C.

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturatingmore » conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.« less

Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase

PubMed Central

Yuan, Ji; Chow, Dar-Chone; Huang, Wanzhi; Palzkill, Timothy

2011-01-01

The β-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits treatment strategies for Staphylococcus infections. The goal of this study was to determine the binding affinity of BLIP for S. aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (Ki) for the PC1 β-lactamase was measured at 350 nM and isothermal titration calorimetry (ITC) experiments indicated a binding constant (Kd) of 380 nM. A total of 23 residue positions in BLIP that contact β-lactamase were randomized and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP K74G mutant was the dominant clone selected and it was found to inhibit the PC1 β-lactamase with a Ki of 42 nM while calorimetry indicated a Kd of 26 nM. Molecular modeling studies suggested BLIP binds weakly to the PC1 β-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme where it forms a salt bridge with BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the A104E PC1 enzyme binds BLIP with 15-fold greater affinity than wild type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with the variation in affinity due to altered dissociation rate constants suggesting changes in short-range interactions are responsible for the altered binding properties of the mutants. PMID:21238457

Dynamic Factors Affecting Gaseous Ligand Binding in an Artificial Oxygen Transport Protein‡

PubMed Central

Zhang, Lei; Andersen, Eskil M.E.; Khajo, Abdelahad; Magliozzo, Richard S.; Koder, Ronald L.

2013-01-01

We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7 this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime which may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when when exposed to oxygen. Compared to HP7, distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off-rate. EPR comparison of these ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation greatly increases water penetration into the protein core. The inability of the mutant protein to bind oxygen may be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together these data underline the importance of the control of protein dynamics in the design of functional artificial proteins. PMID:23249163

Dynamic factors affecting gaseous ligand binding in an artificial oxygen transport protein.

PubMed

Zhang, Lei; Andersen, Eskil M E; Khajo, Abdelahad; Magliozzo, Richard S; Koder, Ronald L

2013-01-22

We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7, this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities, and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime that may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when exposed to oxygen. Compared to that of HP7, the distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off rate. Electron paramagnetic resonance comparison of these ferric hemoproteins demonstrates that the mutation increases the level of disorder at the heme binding site. Nuclear magnetic resonance-detected deuterium exchange demonstrates that the mutation greatly increases the degree of penetration of water into the protein core. The inability of the mutant protein to bind oxygen may be due to an increased level of water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together, these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.

Computational Approaches for Decoding Select Odorant-Olfactory Receptor Interactions Using Mini-Virtual Screening

PubMed Central

Harini, K.; Sowdhamini, Ramanathan

2015-01-01

Olfactory receptors (ORs) belong to the class A G-Protein Coupled Receptor superfamily of proteins. Unlike G-Protein Coupled Receptors, ORs exhibit a combinatorial response to odors/ligands. ORs display an affinity towards a range of odor molecules rather than binding to a specific set of ligands and conversely a single odorant molecule may bind to a number of olfactory receptors with varying affinities. The diversity in odor recognition is linked to the highly variable transmembrane domains of these receptors. The purpose of this study is to decode the odor-olfactory receptor interactions using in silico docking studies. In this study, a ligand (odor molecules) dataset of 125 molecules was used to carry out in silico docking using the GLIDE docking tool (SCHRODINGER Inc Pvt LTD). Previous studies, with smaller datasets of ligands, have shown that orthologous olfactory receptors respond to similarly-tuned ligands, but are dramatically different in their efficacy and potency. Ligand docking results were applied on homologous pairs (with varying sequence identity) of ORs from human and mouse genomes and ligand binding residues and the ligand profile differed among such related olfactory receptor sequences. This study revealed that homologous sequences with high sequence identity need not bind to the same/ similar ligand with a given affinity. A ligand profile has been obtained for each of the 20 receptors in this analysis which will be useful for expression and mutation studies on these receptors. PMID:26221959

Nuclear factor ETF specifically stimulates transcription from promoters without a TATA box.

PubMed

Kageyama, R; Merlino, G T; Pastan, I

1989-09-15

Transcription factor ETF stimulates the expression of the epidermal growth factor receptor (EGFR) gene which does not have a TATA box in the promoter region. Here, we show that ETF recognizes various GC-rich sequences including stretches of deoxycytidine or deoxyguanosine residues and GC boxes with similar affinities. ETF also binds to TATA boxes but with a lower affinity. ETF stimulated in vitro transcription from several promoters without TATA boxes but had little or no effect on TATA box-containing promoters even though they had strong ETF-binding sites. These inactive ETF-binding sites became functional when placed upstream of the EGFR promoter whose own ETF-binding sites were removed. Furthermore, when a TATA box was introduced into the EGFR promoter, the responsiveness to ETF was abolished. These results indicate that ETF is a specific transcription factor for promoters which do not contain TATA elements.

[125I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

PubMed Central

Dumont, Yvan; Quirion, Rémi

2000-01-01

GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]-GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]-GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [125I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]-GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]-GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. PMID:10694200

Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

PubMed

Feuerstein, Sophie E; Pulvermüller, Alexander; Hartmann, Rudolf; Granzin, Joachim; Stoldt, Matthias; Henklein, Peter; Ernst, Oliver P; Heck, Martin; Willbold, Dieter; Koenig, Bernd W

2009-11-17

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.

High Resolution Features from Low Affinity Interactions: Photoactive Analogs of the Haloether Anesthetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xi,J.; Liu, R.; Rossi, M.

2006-01-01

The difficulty in obtaining binding target and site information for low-affinity drugs, like the inhaled anesthetics, has limited identification of their molecular effectors. Because such information can be provided by photoactive analogues, we designed, synthesized, and characterized a novel diazirnyl haloether that closely mimics isoflurane, the most widely used clinical general anesthetic. This compound, H-diaziflurane, is a nontoxic, potent anesthetic that potentiates GABA-gated ion channels in primary cultures of hippocampal neurons. Calorimetric and structural characterizations show that H-diaziflurane binds a model anesthetic host protein with similar energetics as isoflurane and forms photoadducts with residues lining the isoflurane binding site. H-diazifluranemore » will be immediately useful for identifying targets and sites important for the molecular pharmacology of the inhaled haloether anesthetics.« less

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

PubMed

Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

2018-06-01

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

PubMed

Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

2013-12-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity

PubMed Central

Sharma, P.; Postel, S.; Sundberg, E.J.; Kranz, D.M.

2013-01-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

Imaging Neuroinflammation in Post Traumatic Stress Disorder

DTIC Science & Technology

2012-11-01

Metabolite B = 0-30%), without evidence of lipophilic metabolites which can confound the analysis. 8 Figure 2 Left graph : Mean PSTD... graph : There is similar plasma protein binding of 18-F PBR111 in healthy and PTSD participants. Individual subject are data are indicated on the... graph . TSPO Binder status Both mixed and high afffinity TSPO binders were evident in the PTSD (4 high affinity binders, 4 mixed affinity

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

PubMed

Shrestha, Stal S; Liow, Jeih-San; Jenko, Kimberly; Ikawa, Masamichi; Zoghbi, Sami S; Innis, Robert B

2016-12-01

Prazosin, a potent and selective α 1 -adrenoceptor antagonist, displaces 25% of 11 C-CUMI-101 ([O-methyl- 11 C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11 C-CUMI-101 binding to α 1 -adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3 H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3 H-prazosin and the inhibition constant of CUMI-101. α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. Reasoning by analogy, 25% of 11 C-CUMI-101 uptake in human cerebellum reflects binding to α 1 -adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

PubMed

Clark, Andrew G; Naufer, M Nabuan; Westerlund, Fredrik; Lincoln, Per; Rouzina, Ioulia; Paramanathan, Thayaparan; Williams, Mark C

2018-02-06

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

Assessing the binding of cholinesterase inhibitors by docking and molecular dynamics studies.

PubMed

Ali, M Rejwan; Sadoqi, Mostafa; Møller, Simon G; Boutajangout, Allal; Mezei, Mihaly

2017-09-01

In this report we assessed by docking and molecular dynamics the binding mechanisms of three FDA-approved Alzheimer drugs, inhibitors of the enzyme acetylcholinesterase (AChE): donepezil, galantamine and rivastigmine. Dockings by the softwares Autodock-Vina, PatchDock and Plant reproduced the docked conformations of the inhibitor-enzyme complexes within 2Å of RMSD of the X-ray structure. Free-energy scores show strong affinity of the inhibitors for the enzyme binding pocket. Three independent Molecular Dynamics simulation runs indicated general stability of donepezil, galantamine and rivastigmine in their respective enzyme binding pocket (also referred to as gorge) as well as the tendency to form hydrogen bonds with the water molecules. The binding of rivastigmine in the Torpedo California AChE binding pocket is interesting as it eventually undergoes carbamylation and breaks apart according to the X-ray structure of the complex. Similarity search in the ZINC database and targeted docking on the gorge region of the AChE enzyme gave new putative inhibitor molecules with high predicted binding affinity, suitable for potential biophysical and biological assessments. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases.

PubMed

Soya, Naoto; Shoemaker, Glen K; Palcic, Monica M; Klassen, John S

2009-11-01

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5more » (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).« less

Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

PubMed Central

Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.

2013-01-01

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA. PMID:23137296

Boosting Affinity by Correct Ligand Preorganization for the S2 Pocket of Thrombin: A Study by Isothermal Titration Calorimetry, Molecular Dynamics, and High-Resolution Crystal Structures.

PubMed

Rühmann, Eggert H; Rupp, Melinda; Betz, Michael; Heine, Andreas; Klebe, Gerhard

2016-02-04

Structural preorganization to fix bioactive conformations at protein binding sites is a popular strategy to enhance binding affinity during late-stage optimization. The rationale for this enhancement relates to entropic advantages assigned to rigidified versus flexible ligands. We analyzed a narrow series of peptidomimetics binding to thrombin. The individual ligands exhibit at P2 a conformationally flexible glycine, more restricted alanine, N-methylglycine, N-methylhomoalanine, and largely rigidified proline moiety. Overall, affinity was found to increase by a factor of 1000, explained partly by an entropic advantage. All ligands adopt the same binding mode with small deviations. The residual mobility of the bound ligands is decreased across the series, and a protein side chain differs in its order/disorder behavior along with changes in the surface-water network pattern established across the newly generated protein-ligand surfaces. The enthalpy/entropy inventory displays a rather complex picture and emphasizes that thermodynamics can only be compared in terms of relative differences within a structurally similar ligand series. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.H.; el-Fakahany, E.E.

1985-06-01

The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/supmore » 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.« less

AMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding

PubMed Central

Bieri, Michael; Mobbs, Jesse I.; Koay, Ann; Louey, Gavin; Mok, Yee-Foong; Hatters, Danny M.; Park, Jong-Tae; Park, Kwan-Hwa; Neumann, Dietbert; Stapleton, David; Gooley, Paul R.

2012-01-01

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β1 and β2). Muscle-specific β2-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β1-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β2-CBM is concurrent with an increase in flexibility in β2-CBM, which may account for the affinity differences between the two isoforms. In contrast to β1-CBM, unbound β2-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β2-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β2-CBM mutant. Insertion of a threonine into the background of β1-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms. PMID:22339867

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins.

PubMed

Marinelli, Fabrizio; Kuhlmann, Sonja I; Grell, Ernst; Kunte, Hans-Jörg; Ziegler, Christine; Faraldo-Gómez, José D

2011-12-06

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na(+)-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581(T), and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell.

PubMed

Sahu, Santosh Kumar; Aradhyam, Gopala Krishna; Gummadi, Sathyanarayana N

2009-10-01

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca2+ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca2+/Mg2+ binding to human scramblases and conformational changes taking place in them remains unknown. In the present study, we analyzed the Ca2+ and Mg2+ binding to the calcium binding motifs of hPLSCR1-4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry. The results in this study show that (i) affinities of the peptides are in the order hPLSCR1>hPLSCR3>hPLSCR2>hPLSCR4 for Ca2+ and in the order hPLSCR1>hPLSCR2>hPLSCR3>hPLSCR4 for Mg2+, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca2+ and Mg2+ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families. Based on the above results, we hypothesize that the Ca2+ binding motif of hPLSCR1 is a novel type of Ca2+ binding motif. Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.

Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

PubMed Central

Parry, Jesse J.; Chen, Ronald; Andrews, Rebecca; Lears, Kimberly A.

2012-01-01

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling. PMID:22495673

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate

PubMed Central

Volpon, Laurent; Osborne, Michael J.; Zahreddine, Hiba; Romeo, Andrea A.; Borden, Katherine L.B.

2013-01-01

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m7G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5μM eIF4E) but much weaker affinity at more typical NMR concentrations (50-200μM). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central β-sheet and dorsal surface of eIF4E, similar to m7G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in both drug design efforts and reveal new insights into ligand eIF4E recognition. PMID:23583375

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

Computing the binding affinity of a ligand buried deep inside a protein with the hybrid steered molecular dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villarreal, Oscar D.; Yu, Lili; Department of Laboratory Medicine, Yancheng Vocational Institute of Health Sciences, Yancheng, Jiangsu 224006

Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions. These errors propagate to the final results of the computed binding affinities even if we were able to perfectly implement the statistical mechanicsmore » of all the processes relevant to a given problem. And they are actually amplified to various degrees even in the mature, sophisticated computational approaches. In particular, the free energy perturbation (alchemical) approaches amplify the errors in the force field parameters because they rely on extracting the small differences between similarly large numbers. In this paper, we develop a hybrid steered molecular dynamics (hSMD) approach to the difficult binding problems of a ligand buried deep inside a protein. Sampling the transition along a physical (not alchemical) dissociation path of opening up the binding cavity- -pulling out the ligand- -closing back the cavity, we can avoid the problem of error amplifications by not relying on small differences between similar numbers. We tested this new form of hSMD on retinol inside cellular retinol-binding protein 1 and three cases of a ligand (a benzylacetate, a 2-nitrothiophene, and a benzene) inside a T4 lysozyme L99A/M102Q(H) double mutant. In all cases, we obtained binding free energies in close agreement with the experimentally measured values. This indicates that the force field parameters we employed are accurate and that hSMD (a brute force, unsophisticated approach) is free from the problem of error amplification suffered by many sophisticated approaches in the literature.« less

Discovery of high-affinity BCL6-binding peptide and its structure-activity relationship

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Kotaro; Sogabe, Satoshi; Kamada, Yusuke

B cell lymphoma 6 (BCL6) is a transcriptional repressor that interacts with its corepressors BcoR and SMRT. Since this protein-protein interaction (PPI) induces activation and differentiation of B lymphocytes, BCL6 has been an attractive drug target for potential autoimmune disease treatments. Here we report a novel BCL6 inhibitory peptide, F1324 (Ac-LWYTDIRMSWRVP-OH), which we discovered using phage display technology; we also discuss this peptide's structure-activity relationship (SAR). For BCL6(5-129) binding, K{sub D} and IC{sub 50} values of F1324 were 0.57 nM and 1 nM according to the results of an SPR analysis and cell-free ELISA assay, respectively. In contrast, BcoR(Arg498-514Pro) and SMRT(Leu1422-Arg1438) exhibitedmore » relatively weak micromole-order binding to BCL6. Furthermore, Fusion protein AcGFP-F1324 transiently expressed in HEK293T cells inhibited intracellular PPI in cell-based M2H assay. By examination of the truncation and fragmentation of F1324, the C-terminal sequence WRVP, which is similar to the BcoR(509-512) sequence WVVP, was identified as being critical for BCL6 binding. In addition, subsequent single-crystal X-ray diffraction analysis of F1324/BCL6(5-129) complex revealed that the high affinity of F1324 was caused by effective interaction of its side chains while its main chain structure was similar to that of BcoR(Arg498-514Pro). To our knowledge, F1324 is the strongest BCL6-binding peptide yet reported. - Highlights: • F1324 was discovered as 5000-times higher affinity peptide to BCL6 than that of BcoR(R498-P514). • X-ray crystal structure analysis revealed the binding mode. • To our knowledge, F1324 is the strongest BCL6-binding and -inhibition peptide so far.« less

Synthesis and stereospecificity of 4,5-disubstituted oxazolidinone ligands binding to T-box riboswitch RNA.

PubMed

Orac, Crina M; Zhou, Shu; Means, John A; Boehm, David; Bergmeier, Stephen C; Hines, Jennifer V

2011-10-13

The enantiomers and the cis isomers of two previously studied 4,5-disubstituted oxazolidinones have been synthesized, and their binding to the T-box riboswitch antiterminator model RNA has been investigated in detail. Characterization of ligand affinities and binding site localization indicates that there is little stereospecific discrimination for binding antiterminator RNA alone. This binding similarity between enantiomers is likely due to surface binding, which accommodates ligand conformations that result in comparable ligand-antiterminator contacts. These results have significant implications for T-box antiterminator-targeted drug discovery and, in general, for targeting other medicinally relevant RNA that do not present deep binding pockets.

Synthesis and stereospecificity of 4,5-disubstituted oxazolidinone ligands binding to T-box riboswitch RNA

PubMed Central

Orac, Crina M.; Zhou, Shu; Means, John A.; Boehm, David; Bergmeier, Stephen C.; Hines, Jennifer V.

2012-01-01

The enantiomers and the cis isomers of two previously studied 4,5-disubstituted oxazolidinones have been synthesized and their binding to the T-box riboswitch antiterminator model RNA investigated in detail. Characterization of ligand affinities and binding site localization indicate that there is little stereospecific discrimination for binding antiterminator RNA alone. This binding similarity between enantiomers is likely due to surface binding, which accommodates ligand conformations that result in comparable ligand-antiterminator contacts. These results have significant implications for T-box antiterminator-targeted drug discovery and, in general, for targeting other medicinally relevant RNA that do not present deep binding pockets. PMID:21812425

A general and fast scoring function for protein-ligand interactions: a simplified potential approach.

PubMed

Muegge, I; Martin, Y C

1999-03-11

A fast, simplified potential-based approach is presented that estimates the protein-ligand binding affinity based on the given 3D structure of a protein-ligand complex. This general, knowledge-based approach exploits structural information of known protein-ligand complexes extracted from the Brookhaven Protein Data Bank and converts it into distance-dependent Helmholtz free interaction energies of protein-ligand atom pairs (potentials of mean force, PMF). The definition of an appropriate reference state and the introduction of a correction term accounting for the volume taken by the ligand were found to be crucial for deriving the relevant interaction potentials that treat solvation and entropic contributions implicitly. A significant correlation between experimental binding affinities and computed score was found for sets of diverse protein-ligand complexes and for sets of different ligands bound to the same target. For 77 protein-ligand complexes taken from the Brookhaven Protein Data Bank, the calculated score showed a standard deviation from observed binding affinities of 1.8 log Ki units and an R2 value of 0.61. The best results were obtained for the subset of 16 serine protease complexes with a standard deviation of 1.0 log Ki unit and an R2 value of 0.86. A set of 33 inhibitors modeled into a crystal structure of HIV-1 protease yielded a standard deviation of 0.8 log Ki units from measured inhibition constants and an R2 value of 0.74. In contrast to empirical scoring functions that show similar or sometimes better correlation with observed binding affinities, our method does not involve deriving specific parameters that fit the observed binding affinities of protein-ligand complexes of a given training set. We compared the performance of the PMF score, Böhm's score (LUDI), and the SMOG score for eight different test sets of protein-ligand complexes. It was found that for the majority of test sets the PMF score performs best. The strength of the new approach presented here lies in its generality as no knowledge about measured binding affinities is needed to derive atomic interaction potentials. The use of the new scoring function in docking studies is outlined.

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins.

PubMed

Suzuki, N; Mihashi, K

1991-01-01

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.

Toxic metals (Ni2+, Pb2+, Hg2+) binding affinity of dissolved organic matter (DOM) derived from different ages municipal landfill leachate

NASA Astrophysics Data System (ADS)

Rikta, S. Y.; Tareq, Shafi M.; Uddin, M. Khabir

2018-03-01

Solid waste production is rapidly increasing in Bangladesh and landfill leachate is the consequence of the decomposition of this waste. These leachates contain heavy metals and significant amount of dissolved organic matter (DOM). DOM is known to have considerable role in heavy metals speciation. Hence, it is important to characterize DOM/leachate and evaluate toxic metals binding affinity of DOM. The objectives of this study were to characterize the DOM in landfill leachate through physico-chemical and optical analyses and to investigate the toxic metals (Ni2+, Pb2+ and Hg2+) binding affinity of three different ages (fresh sample L-1, young sample L-2 and mature sample L-3) DOM samples. Results suggested that leachate is a potential pollutant which contained very high organic pollutant load. Conditional stability constant (Log K) and percentages of fluorophores that correspond to metal binding (% f) values indicated that young DOM sample (L-2) had the highest binding affinity to all the three metals ions. In general, DOM samples showed the following order affinity to the metal ions; Ni2+ binding affinity: L-2 > L-3 > L-1, Pb2+ binding affinity: L-2 > L-3 > L-1 and Hg2+ binding affinity: L-2 > L-1 > L-3.

Sigma opiates and certain antipsychotic drugs mutually inhibit (+)-[3H] SKF 10,047 and [3H]haloperidol binding in guinea pig brain membranes.

PubMed Central

Tam, S W; Cook, L

1984-01-01

The relationship between binding of antipsychotic drugs and sigma psychotomimetic opiates to binding sites for the sigma agonist (+)-[3H]SKF 10,047 (N-allylnormetazocine) and to dopamine D2 sites was investigated. In guinea pig brain membranes, (+)-[3H]SKF 10,047 bound to a single class of sites with a Kd of 4 X 10(-8) M and a Bmax of 333 fmol/mg of protein. This binding was different from mu, kappa, or delta opiate receptor binding. It was inhibited by opiates that produce psychotomimetic activities but not by opiates that lack such activities. Some antipsychotic drugs inhibited (+)-[3H]SKF 10,047 binding with high to moderate affinities in the following order of potency: haloperidol greater than perphenazine greater than fluphenazine greater than acetophenazine greater than trifluoperazine greater than molindone greater than or equal to pimozide greater than or equal to thioridazine greater than or equal to chlorpromazine greater than or equal to triflupromazine. However, there were other antipsychotic drugs such as spiperone and clozapine that showed low affinity for the (+)-[3H]SKF 10,047 binding sites. Affinities of antipsychotic drugs for (+)-[3H]SKF 10,047 binding sites did not correlate with those for [3H]spiperone (dopamine D2) sites. [3H]-Haloperidol binding in whole brain membranes was also inhibited by the sigma opiates pentazocine, cyclazocine, and (+)-SKF 10,047. In the striatum, about half of the saturable [3H]haloperidol binding was to [3H]spiperone (D2) sites and the other half was to sites similar to (+)-[3H]SKF 10,047 binding sites. PMID:6147851

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

PubMed Central

Altman, Michael D.; Nalivaika, Ellen A.; Prabu-Jeyabalan, Moses; Schiffer, Celia A.; Tidor, Bruce

2009-01-01

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods — namely, charge optimization and protein design — were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT–RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a ten-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease. PMID:17729291

An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

PubMed

Shoots, Jenny; Fraccalvieri, Domenico; Franks, Diana G; Denison, Michael S; Hahn, Mark E; Bonati, Laura; Powell, Wade H

2015-06-02

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1β (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

PubMed Central

Naveau, Marie; Lazennec-Schurdevin, Christine; Panvert, Michel; Dubiez, Etienne; Mechulam, Yves; Schmitt, Emmanuelle

2013-01-01

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex. PMID:23193270

Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

DOE PAGES

Lin, Yen -Lin; Meng, Yilin; Huang, Lei; ...

2014-10-22

Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to themore » DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.« less

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

2014-01-01

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

PubMed

Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

2014-01-01

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum.

PubMed

Liebau, Eva; De Maria, Francesca; Burmeister, Cora; Perbandt, Markus; Turella, Paola; Antonini, Giovanni; Federici, Giorgio; Giansanti, Francesco; Stella, Lorenzo; Lo Bello, Mario; Caccuri, Anna Maria; Ricci, Giorgio

2005-07-15

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Neurochemical correlates of. gamma. -aminobutyrate (GABA) inhibition in cat visual cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balcar, V.J.; Dreher, B.

1990-01-01

High affinity binding of ({sup 3}H){gamma}-aminobutyric acid (GABA) to neuronal membranes from different parts of cat visual cortex was tested for sensitivity to GABA{sub A} agonists isoguvacine and THIP, GABA{sub A} antagonist SR95531 and GABA{sub B} agonist baclofen. Some of the GABA{sub A}-binding sites were found to have a very low affinity for THIP, suggesting the presence and, possibly, uneven distribution of non-synaptic GABA{sub A} receptors in cat visual cortex. There were no differences in K{sub m} and V{sub max} values of high affinity uptake of GABA and in the potency of K{sup +}-stimulated release of GABA, between primary andmore » association cortices. Consequently, the present results indicate that despite the anatomical and physiological differences between the primary and association feline visual cortices the neurochemical characteristics of GABAergic inhibition are very similar in the two regions.« less

[Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

PubMed

Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

2013-05-01

A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

RNA aptamers that functionally interact with green fluorescent protein and its derivatives

PubMed Central

Shui, Bo; Ozer, Abdullah; Zipfel, Warren; Sahu, Nevedita; Singh, Avtar; Lis, John T.; Shi, Hua; Kotlikoff, Michael I.

2012-01-01

Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways. PMID:22189104

Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

PubMed

Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

2016-04-01

The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

Use of receptor chimeras to identify small molecules with high affinity for the dynorphin A binding domain of the kappa opioid receptor.

PubMed

Kumar, Virendra; Guo, Deqi; Marella, Michael; Cassel, Joel A; Dehaven, Robert N; Daubert, Jeffrey D; Mansson, Erik

2008-06-15

A series of 2-substituted sulfamoyl arylacetamides of general structure 2 were prepared as potent kappa opioid receptor agonists and the affinities of these compounds for opioid and chimeric receptors were compared with those of dynorphin A. Compounds 2e and 2i were identified as non-peptide small molecules that bound to chimeras 3 and 4 with high affinities similar to dynorphin A, resulting in K(i) values of 1.5 and 1.2 nM and 1.3 and 2.2 nM, respectively.

Is There Consistency between the Binding Affinity and Inhibitory Potential of Natural Polyphenols as α-amylase Inhibitors?

PubMed

Xu, Wei; Shao, Rong; Xiao, Jianbo

2016-07-26

The inhibitory potential of natural polyphenols for α-amylases has attracted great interests among researchers. The structure-affinity properties of natural polyphenols binding to α-amylase and the structure-activity relationship of dietary polyphenols inhibiting α-amylase were deeply investigated. There is a lack of consistency between the structure-affinity relationship and the structure-activity relationship of natural polyphenols as α-amylase inhibitors. Is it consistent between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors? It was found that the consistency between the binding affinity and inhibitory potential of natural polyphenols as with α-amylase inhibitors is not equivocal. For example, there is no consistency between the binding affinity and the inhibitory potential of quercetin and its glycosides as α-amylase inhibitors. However, catechins with higher α-amylase inhibitory potential exhibited higher affinity with α-amylase.

Introduction of structural affinity handles as a tool in selective nucleic acid separations

NASA Technical Reports Server (NTRS)

Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

2011-01-01

The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids.

PubMed

Torres, Oscar B; Duval, Alexander J; Sulima, Agnieszka; Antoline, Joshua F G; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

2018-06-01

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

Bone sialoprotein-collagen interaction promotes hydroxyapatite nucleation.

PubMed

Baht, Gurpreet S; Hunter, Graeme K; Goldberg, Harvey A

2008-09-01

In bone, hydroxyapatite (HA) crystals are deposited onto the type I collagen scaffold by a mechanism that has yet to be elucidated. Bone sialoprotein (BSP) is an acidic phosphoprotein that is expressed at high levels in mineralized tissues, capable of binding type I collagen, and nucleating HA. Both bone-extracted and recombinant BSP (rBSP) bind with equal affinity to collagen. The nature of the BSP-collagen interaction and its role in HA nucleation are not known. We have used a solid-phase binding assay and affinity chromatography to characterize the BSP-collagen interaction. rBSP-binding affinities of triple-helical and fibrillar type I collagen were similar (K(D) approximately 13 nM), while that of heat-denatured type I collagen was lower (K(D) approximately 44 nM), indicating the importance of triple-helical structure in binding BSP. Pepsin treatment of collagen had no effect on rBSP binding, demonstrating that the telopeptides of collagen are not involved. The majority of collagen-bound rBSP was eluted by acetonitrile, indicating that hydrophobic interactions are principally responsible for binding. Using an HA-nucleation assay, it was shown that rBSP is ten-fold more potent in reconstituted fibrillar collagen gels than in agarose gels. Nucleating potency of a non-collagen-binding, HA-nucleating peptide [rBSP(134-206)] showed no difference in the two gel systems. The work here shows that optimal binding of rBSP requires collagen to be in a native, triple-helical structure, does not require the telopeptides, and is stabilized by hydrophobic interactions. Upon binding to collagen, rBSP displays an increase in nucleation potency, implying a co-operative effect of BSP and collagen in mineral formation.

sigma opiates and certain antipsychotic drugs mutually inhibit (+)-(/sup 3/H)SKF 10,047 and (/sup 3/H)haloperidol binding in guinea pig brain membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tam, S.W.; Cook, L.

1984-09-01

The relationship between binding of antipsychotic drugs and sigma psychotomimetic opiates to binding sites for the sigma agonist (+)-(/sup 3/H)SKF 10,047 (N-allylnormetazocine) and to dopamine D/sub 2/ sites was investigated. In guinea pig brain membranes, (+)-(/sup 3/H)SKF 10,047 bound to single class of sites with a K/sub d/ of 4 x 10/sup -8/ M and a B/sub max/ of 333 fmol/mg of protein. This binding was different from ..mu.., kappa, or delta opiate receptor binding. It was inhibited by opiates that produce psychotomimetic activities but not by opiates that lack such activities. Some antipsychotic drugs inhibited (+)-(/sup 3/H)SKF 10,047 bindingmore » with high to moderate affinities in the following order of potency: haloperidol > perphenazine > fluphenazine > acetophenazine > trifluoperazine > molindone greater than or equal to pimozide greater than or equal to thioridazine greater than or equal to chlorpromazine greater than or equal to triflupromazine. However, there were other antipsychotic drugs such as spiperone and clozapine that showed low affinity for the (+)-(/sup 3/H)SKF 10,047 binding sites. Affinities of antipsychotic drugs for (+)-(/sup 3/H)SKF 10,047 binding sites did not correlate with those for (/sup 3/H)spiperone (dopamine D/sub 2/) sites. (/sup 3/H)-Haloperidol binding in whole brain membranes was also inhibited by the sigma opiates pentazocine, cyclazocine, and (+)-(/sup 3/H)SKF 10,047. In the striatum, about half of the saturable (/sup 3/H)haloperidol binding was to (/sup 3/H)spiperone (D/sub 2/) sites and the other half was to sites similar to (+)-(/sup 3/H)SKF 10,047 binding sites. 15 references, 4 figures, 1 table.« less

Comparison of Nerve Growth Factor Receptor Binding Models Using Heterodimeric Muteins

PubMed Central

Mehta, Hrishikesh M.; Woo, Sang B.; Neet, Kenneth E.

2013-01-01

Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA. PMID:22903500

Characterization of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

PubMed

Talkad, V D; Patto, R J; Metz, D C; Turner, R J; Fortune, K P; Bhat, S T; Gardner, J D

1994-10-20

By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy the low affinity state of the CCK receptor. When amplification was expressed quantitatively as the value of Kd for the low affinity state divided by the corresponding EC50 for stimulating amylase secretion the values were CCK-8 (1000), de(SO)CCK-8 (1500), gastrin (100) and CCK-JMV-180 (Menozzi, D., Vinayek, R., Jensen, R.T. and Gardner, J.D. (1991) J. Biol. Chem. 266, 10385-1091).(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

PubMed

Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

2012-10-15

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

Existence of three subtypes of bradykinin B2 receptors in guinea pig.

PubMed

Seguin, L; Widdowson, P S; Giesen-Crouse, E

1992-12-01

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)

(3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Norman, A.B.; Battaglia, G.; Creese, I.

1985-12-01

In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

Two-dimensional Kinetics Regulation of αLβ2-ICAM-1 Interaction by Conformational Changes of the αL-Inserted Domain*

PubMed Central

Zhang, Fang; Marcus, Warren D.; Goyal, Nimita H.; Selvaraj, Periasamy; Springer, Timothy A.; Zhu, Cheng

2006-01-01

The leukocyte integrin αLβ2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of αLβ2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with αLβ2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by ~8000- and ~30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type αLβ2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the αLβ2, is required for affinity and on-rate up-regulation. PMID:16234238

Characterization of binding affinity of CJ-023,423 for human prostanoid EP4 receptor.

PubMed

Murase, Akio; Nakao, Kazunari; Takada, Junji

2008-01-01

In order to characterize the receptor binding pharmacology of CJ-023,423, a potent and selective EP4 antagonist, we performed a radioligand receptor binding assay under various assay conditions. An acidic (pH 6) and hypotonic buffer is a conventional, well-known buffer for prostaglandin E2 receptor binding assays. CJ-023,423 showed moderate binding affinity for human EP4 receptor under conventional buffer conditions. However, its binding affinity was greatly increased under neutral (pH 7.4) and isotonic buffer conditions. In this report, the binding mechanism between CJ-023,423 and human EP4 receptor is discussed based on the binding affinities determined under various assay conditions. Copyright 2008 S. Karger AG, Basel.

High-aluminum-affinity silica is a nanoparticle that seeds secondary aluminosilicate formation.

PubMed

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m(2) g(-1) and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates.

High-Aluminum-Affinity Silica Is a Nanoparticle That Seeds Secondary Aluminosilicate Formation

PubMed Central

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J.

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m2 g-1 and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates. PMID:24349573

Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction.

PubMed

Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl; Hejazi, Mohammad Saeid

2014-01-01

Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA), raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting "A site" of 16S rRNA) to riboswitches via docking method. There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8) hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with "16S rRNA A site". Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA "A site" suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Torque Generation Mechanism of F1-ATPase upon NTP Binding

PubMed Central

Arai, Hidenobu C.; Yukawa, Ayako; Iwatate, Ryu John; Kamiya, Mako; Watanabe, Rikiya; Urano, Yasuteru; Noji, Hiroyuki

2014-01-01

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F1-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation. Although the binding rates of UTP and ribose triphosphate were 103 and 106 times, respectively, slower than that of ATP, they supported rotation, generating torque comparable to that generated by ATP. Affinity change of F1 to UTP coupled with rotation was determined, and the results again were comparable to those for ATP, suggesting that F1 exerts torque upon the affinity change to UTP via rotation similar to ATP-driven rotation. Thus, the adenine-ring significantly enhances the binding rate, although it is not directly involved in force generation. Taking into account the findings from another study on F1 with mutated phosphate-binding residues, it was proposed that progressive bond formation between the phosphate region and catalytic residues is responsible for the rotation-coupled change in affinity. PMID:24988350

Revealing multi-binding sites for taspine to VEGFR-2 by cell membrane chromatography zonal elution.

PubMed

Du, Hui; Wang, Sicen; Ren, Jing; Lv, Nan; He, Langchong

2012-03-01

A new high-expression vascular endothelial growth factor receptor-2 (VEGFR-2) cell membrane chromatography (CMC) method was developed to investigate the affinity of ligands for VEGFR-2. An HEK293 VEGFR-2/CMC system was applied to specifically recognize ligands acting on VEGFR-2. Sorafenib was used as a mobile phase additive to evaluate the effect of the marker's concentration on the retention of sorafenib and taspine, respectively. The relationship among the retention, the types of binding sites and the affinity of taspine binding to VEGFR-2 has also been concerned. The retention behavior indicated that sorafenib had two major binding regions on VEGFR-2, and that taspine might act as a multi-target VEGFR-2 inhibitor with similar biological activity to sorafenib. The equilibrium dissociation constants (K(D)) obtained from the model are (5.25 ± 0.31) × 10⁻⁷ and (9.88 ± 0.54) × 10⁻⁵ mol L⁻¹ for sorafenib at the high- and low-affinity sites, respectively, and the corresponding values for taspine are (3.88 ± 0.31) × 10⁻⁶ and (7.04 ± 0.49)×10⁻⁵ mol L⁻¹. The two types of binding sites contributed about a 1:2 ratio on the retention of taspine. Copyright © 2012 Elsevier B.V. All rights reserved.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

SAMPL4 & DOCK3.7: lessons for automated docking procedures

NASA Astrophysics Data System (ADS)

Coleman, Ryan G.; Sterling, Teague; Weiss, Dahlia R.

2014-03-01

The SAMPL4 challenges were used to test current automated methods for solvation energy, virtual screening, pose and affinity prediction of the molecular docking pipeline DOCK 3.7. Additionally, first-order models of binding affinity were proposed as milestones for any method predicting binding affinity. Several important discoveries about the molecular docking software were made during the challenge: (1) Solvation energies of ligands were five-fold worse than any other method used in SAMPL4, including methods that were similarly fast, (2) HIV Integrase is a challenging target, but automated docking on the correct allosteric site performed well in terms of virtual screening and pose prediction (compared to other methods) but affinity prediction, as expected, was very poor, (3) Molecular docking grid sizes can be very important, serious errors were discovered with default settings that have been adjusted for all future work. Overall, lessons from SAMPL4 suggest many changes to molecular docking tools, not just DOCK 3.7, that could improve the state of the art. Future difficulties and projects will be discussed.

The sodium channel in membranes of electroplax. Binding of batrachotoxinin-a [(3)H]benzoate to particulate preparations from electric eel (electrophorus).

PubMed

McNeal, E T; Daly, J W

1986-01-01

Batrachotoxinin-A [(3)H]benzoate ([(3)H]BTX-B) binds specifically and with high affinity (K(D) 48 nM) to sites (B(max) 2.1 pmol/mg protein) associated with voltage-dependent sodium channels in rodent brain vesicular preparations. High affinity binding requires the presence of scorpion (Leiurus) venom and a membrane potential. Local anesthetics antagonize the binding. Nonspecific binding is defined in the presence of veratridine. In particulate preparations from electroplax of the eel Electrophorus electricus, [(3)H]BTX-B binds with a K(D) of about 140 nM and a B(max) of 2.5 pmol/mg protein in the presence of scorpion venom. Higher concentrations of scorpion venom are required to enhance binding in Electrophorus preparations than in brain preparations. Local anesthetics antagonize binding in Electrophorus preparations with potencies similar to those in brain preparations. Veratridine and batrachotoxin are less potent in blocking binding in Electrophorus than in brain preparations. It appears likely that binding in Electrophorus preparations is primarily to membrane fragments rather than vesicular entities as in brain. Binding of [(3)H]BTX-B to particulate preparations from electroplax of the ray Torpedo californica and the catfish Malapterurus electricus is mainly nonspecific. Scorpion venom does not enhance total binding and local anesthetics are not effective in antagonizing binding.

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

Different roles suggested by sex-biased expression and pheromone binding affinity among three pheromone binding proteins in the pink rice borer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae).

PubMed

Jin, Jun-Yan; Li, Zhao-Qun; Zhang, Ya-Nan; Liu, Nai-Yong; Dong, Shuang-Lin

2014-07-01

Pheromone binding proteins (PBPs) are thought to bind and transport hydrophobic sex pheromone molecules across the aqueous sensillar lymph to specific pheromone receptors on the dendritic membrane of olfactory neurons. A maximum of 3 PBP genes have been consistently identified in noctuid species, and each of them shares high identity with its counterparts in other species within the family. The functionality differences of the 3 proteins are poorly understood. In the present study, 3 PBP cDNAs (SinfPBP1, 2, 3) were identified from the pink rice borer, Sesamia inferens, for the first time. The quantitative real-time PCR indicated that the 3 PBPs displayed similar temporal but very different sex related expression profiles. Expression of SinfPBP1 and SinfPBP2 were highly and moderately male biased, respectively, while SinfPBP3 was slightly female biased, as SinfPBPs were expressed at very different levels (PBP1>PBP2≫PBP3) in male antennae, but at similar levels in female antennae. Furthermore, the 3 SinfPBPs displayed different ligand binding profiles in fluorescence competitive binding assays. SinfPBP1 exhibited high and similar binding affinities to all 3 sex pheromone components (Ki=0.72-1.60 μM), while SinfPBP2 showed selective binding to the alcohol and aldehyde components (Ki=0.78-1.71 μM), and SinfPBP3 showed no obvious binding to the 3 sex pheromone components. The results suggest that SinfPBP1 plays a major role in the reception of female sex pheromones in S. inferens, while SinfPBP3 plays a least role (if any) and SinfPBP2 functions as a recognizer of alcohol and aldehyde components. Copyright © 2014 Elsevier Ltd. All rights reserved.

Validation of tautomeric and protomeric binding modes by free energy calculations. A case study for the structure based optimization of D-amino acid oxidase inhibitors.

PubMed

Orgován, Zoltán; Ferenczy, György G; Steinbrecher, Thomas; Szilágyi, Bence; Bajusz, Dávid; Keserű, György M

2018-02-01

Optimization of fragment size D-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

Validation of tautomeric and protomeric binding modes by free energy calculations. A case study for the structure based optimization of d-amino acid oxidase inhibitors

NASA Astrophysics Data System (ADS)

Orgován, Zoltán; Ferenczy, György G.; Steinbrecher, Thomas; Szilágyi, Bence; Bajusz, Dávid; Keserű, György M.

2018-02-01

Optimization of fragment size d-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors.

PubMed

Xu, Liping; Vagner, Josef; Alleti, Ramesh; Rao, Venkataramanarao; Jagadish, Bhumasamudram; Morse, David L; Hruby, Victor J; Gillies, Robert J; Mash, Eugene A

2010-04-15

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low microM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH(2), exhibited a K(d) for hMC4R of 9.1+/-1.4 microM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-alpha-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects. Copyright 2010 Elsevier Ltd. All rights reserved.

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

PubMed Central

KISSELEVA, NATALIA; KHVOROVA, ANASTASIA; WESTHOF, ERIC; SCHIEMANN, OLAV

2005-01-01

Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of ≤10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G · A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop–loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding. PMID:15611296

Electrostatic Similarity Analysis of Human β-Defensin Binding in the Melanocortin System

PubMed Central

Nix, Matthew A.; Kaelin, Christopher B.; Palomino, Rafael; Miller, Jillian L.; Barsh, Gregory S.; Millhauser, Glenn L.

2015-01-01

Summary The β-defensins are a class of small cationic proteins that serve as components of numerous systems in vertebrate biology, including the immune and melanocortin systems. Human β-defensin 3 (HBD3), which is produced in the skin, has been found to bind to melanocortin receptors 1 and 4 through complementary electrostatics, a unique mechanism of ligand-receptor interaction. This finding indicates that electrostatics alone, and not specific amino acid contact points, could be sufficient for function in this ligand-receptor system, and further suggests that other small peptide ligands could interact with these receptors in a similar fashion. Here, we conducted molecular-similarity analyses and functional studies of additional members of the human β-defensin family, examining their potential as ligands of melanocortin-1 receptor, through selection based on their electrostatic similarity to HBD3. Using Poisson-Boltzmann electrostatic calculations and molecular-similarity analysis, we identified members of the human β-defensin family that are both similar and dissimilar to HBD3 in terms of electrostatic potential. Synthesis and functional testing of a subset of these β-defensins showed that peptides with an HBD3-like electrostatic character bound to melanocortin receptors with high affinity, whereas those that were anticorrelated to HBD3 showed no binding affinity. These findings expand on the central role of electrostatics in the control of this ligand-receptor system and further demonstrate the utility of employing molecular-similarity analysis. Additionally, we identified several new potential ligands of melanocortin-1 receptor, which may have implications for our understanding of the role defensins play in melanocortin physiology. PMID:26536271

Synthesis of biotinylated glycoconjugates and their use in a novel ELISA for direct comparison of HIV-1 Gp120 recognition of GalCer and related carbohydrate analogues.

PubMed

McReynolds, K D; Hadd, M J; Gervay-Hague, J

1999-01-01

As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

PubMed Central

Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

2012-01-01

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

Characterization of (/sup 3/H)forskolin binding sites in the iris-ciliary body of the albino rabbit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldman, M.E.; Mallorga, P.; Pettibone, D.J.

1988-01-01

(/sup 3/H)forskolin binding sites were identified using membranes prepared from the iris-ciliary body of adult, albino rabbits. Scatchard analysis of saturation binding experiments demonstrated that (/sup 3/H)forskolin bound to a single population of high affinity sites. The K/sub d/ and B/sub max/ values were 8.7 +- 0.9 nM and 119.0 +- 30.9 fmolmg prot. using membranes prepared from frozen tissue and 17.0 +- 6.2 nM and 184.4 +- 47.2 fmolmg prot. using fresh tissue. The binding of (/sup 3/H)forskolin was magnesium-dependent. The B/sub max/ was enhanced by sodium fluoride and Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog. Forskolin was the mostmore » potent inhibitor of (/sup 3/H)forskolin binding; two commercially-available analogs were weaker inhibitors. In an adenylate cyclase assay, there was the same rank order of potency to enhance enzyme activity. Based upon binding affinities, magnesium-dependence, sensitivity to sodium fluoride and Gpp(NH)p, rank order of potencies of analogs and correlation of binding with adenylate cyclase activity, these studies suggest that the (/sup 3/H)forskolin binding site in the iris-ciliary body is similar to the binding site in other tissues« less

A study of the uptake of chloroquine in malaria-infected erythrocytes. High and low affinity uptake and the influence of glucose and its analogues.

PubMed

Diribe, C O; Warhurst, D C

1985-09-01

A study of concentration- and substrate-dependence of chloroquine uptake has been carried out on mouse erythrocytes infected with the chloroquine-sensitive NK65 and the chloroquine-resistant RC strains of Plasmodium berghei. The presence of drug binding sites of high and low affinity in such strains of P. berghei was confirmed. High affinity uptake sites in cells parasitized with chloroquine-sensitive and chloroquine-resistant parasites have similar characteristics, but in the sensitive strain the major component of chloroquine-uptake is at high affinity and dependent on the availability of ATP whilst in the resistant strain the major component of uptake is at low affinity and independent of energy. An absolute increase in the quantity of the low affinity site in erythrocytes parasitized with chloroquine-resistant P. berghei was noted, which may be related to an increase in quantity of parasite membrane.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing.

PubMed

Morozov, Giora I; Zhao, Huaying; Mage, Michael G; Boyd, Lisa F; Jiang, Jiansheng; Dolan, Michael A; Venna, Ramesh; Norcross, Michael A; McMurtrey, Curtis P; Hildebrand, William; Schuck, Peter; Natarajan, Kannan; Margulies, David H

2016-02-23

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8(+) T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

DOE PAGES

Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.; ...

2016-02-11

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8 + T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities ofmore » TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.« less

[Interaction of human factor X with thromboplastin].

PubMed

Kiselev, S V; Zubairov, D M; Timarbaev, V N

2003-01-01

The binding of 125I-labeled human factor X to native and papaine-treated tissue tromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard analysis suggests the existence of high (Kd=l,8 x10(-9) M) and low affinity binding sites on the thromboplastin surface. The removal of Ca2+ reduced affinity of factor X to the high affinity sites. This was accompanied by some increase of their number. Proteolysis by papaine decreased affinity of high affinity sites and caused the increase of their number in the presence of Ca2+. In the absence of Ca2+ the affinity remained unchanged, but the number of sites decreased. At low concentrations of factor X positive cooperativity for high affinity binding sites was observed. It did not depend on the presence of Ca2+. The results indirectly confirm the role of hydrophobic interactons in Ca2+ dependent binding of factor X to thromboplastin and the fact that heterogeneity of this binding is determined by mesophase structure of the thromboplastin phospholipids.

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

PubMed Central

Novakovic, Valerie A.; Shi, Jialan; Rasmussen, Jan; Pipe, Steven W.

2015-01-01

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites. PMID:26162408

Copper and the oxidation of hemoglobin: a comparison of horse and human hemoglobins.

PubMed

Rifkind, J M; Lauer, L D; Chiang, S C; Li, N C

1976-11-30

Oxidation studies of hemoglobin by Cu(II) indicate that for horse hemoglobin, up to a Cu(II)/heme molar ratio of 0.5, all of the Cu(II) added is used to rapidly oxidize the heme. On the other hand, most of the Cu(II) added to human hemoglobin at low Cu(II)/heme molar ratios is unable to oxidize the heme. Only at Cu(II)/heme molar ratios greater than 0.5 does the amount of oxidation per added Cu(II) approach that of horse hemoglobin. At the same time, binding studies indicate that human hemoglobin has an additional binding site involving one copper for every two hemes, which has a higher copper affinity than the single horse hemoglobin binding site. The Cu(II) oxidation of human hemoglobin is explained utilizing this additional binding site by a mechanism where a transfer of electrons cannot occur between the heme and the Cu(II) bound to the high affinity human binding site. The electron transfer must involve the Cu(II) bound to the lower affinity human hemoglobin binding site, which is similar to the only horse hemoglobin site. The involvement of beta-2 histidine in the binding of this additional copper is indicated by a comparison of the amino acid sequences of various hemoglobins which possess the additional site, with the amino acid sequences of hemoglobins which do not possess the additional site. Zn(II), Hg(II), and N-ethylmaleimide (NEM) are found to decrease the Cu(II) oxidation of hemoglobin. The sulfhydryl reagents, Hg(II) and NEM, produce a very dramatic decrease in the rate of oxidation, which can only be explained by an effect on the rate for the actual transfer of electrons between the Cu(II) and the Fe(II). The effect of Zn(II) is much smaller and can, for the most part, be explained by the increased oxygen affinity, which affects the ligand dissociation process that must precede the electron transfer process.

“Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

PubMed Central

Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

2015-01-01

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy. PMID:25837251

Allosteric Effects of the Anti-Psychotic Drug Trifluoperazine on the Energetics of Calcium Binding by Calmodulin

PubMed Central

Feldkamp, Michael D.; O'Donnell, Susan E.; Yu, Liping; Shea, Madeline A.

2010-01-01

Trifluoperazine (TFP; Stelazine™) is an antagonist of calmodulin (CaM), an essential regulator of calcium-dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, 1LIN) show TFP bound to (Ca2+)4-CaM in ratios of 1, 2 or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM-kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca2+)4-CaM, and explore differential effects on the N- and C-domains of CaM, stoichiometric TFP titrations of CaM were monitored by 15N-HSQC NMR. Two TFP bound to apo CaM, while four bound to (Ca2+)4-CaM. In both cases, the preferred site was in the C-domain. During the titrations, biphasic responses for some resonances suggested inter-site interactions. TFP-binding sites in apo CaM appeared distinct from those in (Ca2+)4-CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ-motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N-domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed”, “semi-open” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca2+)4-CaM, needs to be considered a potential target of drug action. PMID:20544963

Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuziemko, G.M.; Stroh, M.; Stevens, R.C.

1996-05-21

The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 {times} 10{sup {minus}12} M for GM1 to 1.88 {times} 10{sup {minus}10} M for asialo GM1. The picomolar values obtained by surface plasmon resonance aremore » similar to K{sub d} values determined with whole-cell binding assays. Both whole-cell assays ans SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface. 34 refs., 8 figs., 2 tabs.« less

A Virtual Screening Approach for the Identification of High Affinity Small Molecules Targeting BCR-ABL1 Inhibitors for the Treatment of Chronic Myeloid Leukemia.

PubMed

Sharda, Saphy; Sarmandal, Palash; Cherukommu, Shirisha; Dindhoria, Kiran; Yadav, Manisha; Bandaru, Srinivas; Sharma, Anudeep; Sakhi, Aditi; Vyas, Tanmay; Hussain, Tajamul; Nayarisseri, Anuraj; Singh, Sanjeev Kumar

2017-01-01

CML originates due to reciprocal translocation in Philadelphia chromosome leading to the formation of fusion product BCR-ABL which constitutively activates tyrosine kinase signaling pathways eventually leading to abnormal proliferation of granulocytic cells. As a therapeutic strategy, BCR-ABL inhibitors have been clinically approved which terminates its phosphorylation activity and retards cancer progression. However, a number of patients develop resistance to inhibitors which demand for the discovery of new inhibitors. Given the drawbacks of present inhibitors, by high throughput virtual screening approaches, present study pursues to identify high affinity compounds targeting BCR-ABL1 anticipated to have safer pharmacological profiles. Five established BCR-ABL inhibitors formed the query compounds for identification of structurally similar compounds by Tanimoto coefficient based linear fingerprint search with a threshold of 95% against PubChemdatabase. Assisted by MolDock algorithm all compounds were docked against BCR-ABL protein in order to retrieve high affinity compounds. The parents and similars were further tested for their ADMET propertiesand bioactivity. Rebastinib formed higher affinity inhibitor than rest of the four established compound investigated in the study. Interestingly, Rebastinib similar compound with Pubchem ID: 67254402 was also shown to have highest affinity than other similars including the similars of respective five parents. In terms of ADMET properties Pubchem ID: 67254402 had appreciable ADMET profile and bioactivity. However, Rebastinib still stood as the best inhibitor in terms of binding affinity and ADMET properties than Pubchem ID: 67254402. Nevertheless, owing to the similar pharmacological properties with Rebastinib, Pubchem ID: 67254402 can be expected to form potential BCR-ABL inhibitor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

PubMed

Duggin, Iain G; Matthews, Jacqueline M; Dixon, Nicholas E; Wake, R Gerry; Mackay, Joel P

2005-04-01

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

PubMed Central

Gao, Rong

2015-01-01

ABSTRACT Understanding cellular responses to environmental stimuli requires not only the knowledge of specific regulatory components but also the quantitative characterization of the magnitude and timing of regulatory events. The two-component system is one of the major prokaryotic signaling schemes and is the focus of extensive interest in quantitative modeling and investigation of signaling dynamics. Here we report how the binding affinity of the PhoB two-component response regulator (RR) to target promoters impacts the level and timing of expression of PhoB-regulated genes. Information content has often been used to assess the degree of conservation for transcription factor (TF)-binding sites. We show that increasing the information content of PhoB-binding sites in designed phoA promoters increased the binding affinity and that the binding affinity and concentration of phosphorylated PhoB (PhoB~P) together dictate the level and timing of expression of phoA promoter variants. For various PhoB-regulated promoters with distinct promoter architectures, expression levels appear not to be correlated with TF-binding affinities, in contrast to the intuitive and oversimplified assumption that promoters with higher affinity for a TF tend to have higher expression levels. However, the expression timing of the core set of PhoB-regulated genes correlates well with the binding affinity of PhoB~P to individual promoters and the temporal hierarchy of gene expression appears to be related to the function of gene products during the phosphate starvation response. Modulation of the information content and binding affinity of TF-binding sites may be a common strategy for temporal programming of the expression profile of RR-regulated genes. PMID:26015501

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

Evolution and Protein Packaging of Small Molecule RNA Aptamers

PubMed Central

Lau, Jolene L.; Baksh, Michael M.; Fiedler, Jason D.; Brown, Steven D.; Kussrow, Amanda; Bornhop, Darryl J.; Ordoukhanian, Phillip

2011-01-01

A high-affinity RNA aptamer (Kd = 50 nM) was efficiently identified by SELEX against a heteroaryl dihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (non-encapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique. PMID:21899290

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnes, N.M.; Costall, B.; Egli, P.

The angiotensin converting enzyme (ACE) inhibitor ({sup 3}H)SQ29,852 identified a single high affinity recognition site (defined by 10.0 microM captopril) in the human temporal cortex (pKD 8.62 +/- 0.03; Bmax 248 +/- 24 fmol mg-1 protein, mean +/- S.E.M., n = 4). ACE inhibitors and thiorphan competed to a similar level for the ({sup 3}H)SQ29,852 binding site in the human temporal cortex with a rank order of affinity (pKi values mean +/- S.E.M., n = 3), lisinopril (9.49 +/- 0.02), captopril (9.16 +/- 0.08), SQ29,852 (8.58 +/- 0.04), epicaptopril (7.09 +/- 0.08), fosinopril (7.08 +/- 0.05) and thiorphan (6.40 +/-more » 0.04). Since this rank order of affinity is similar to the affinity of these compounds to inhibit brain ACE activity it is concluded that ({sup 3}H)SQ29,852 selectively labels the inhibitor recognition site of ACE in the human temporal cortex.« less

Hyaluronate-binding proteins of murine brain.

PubMed

Marks, M S; Chi-Rosso, G; Toole, B P

1990-01-01

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Mapping the UDP-Glucuronic Acid Binding Site in UDP-Glucuronosyltransferase-1 A10 by Homology-based Modeling: Confirmation with Biochemical Evidence†

PubMed Central

Banerjee, Rajat; Pennington, Matthew W.; Garza, Amanda; Owens, Ida S.

2008-01-01

The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP-moiety in substrates, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315 and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E /G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of affinity-ligand, 5-azido-uridine-[β-32P]-diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high and low affinity binding sites. 2-Nitro 5-thiocyanobenzoic acid-digested UGT1A10-His bound with radiolabeled affinity-ligand revealed an 11.3- and 14.3-kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater- and 50% less-label in the 14.3-kDa peptide, respectively, compared to 1A10-His without affecting the 11.3-kDa peptide. Scatchard analysis of binding data of affinity-ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in Kd, respectively. Our results indicate: K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high affinity sites and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321--equivalent to K314-- in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDPglcA binding to K314 and K404 in UGT1A10. PMID:18570380

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

PubMed Central

Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

1999-01-01

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: MODEL II

EPA Science Inventory

The training set used to derive a common reactivity pattern (COREPA) model for estrogen receptor (ER) binding affinity in Model I (see Abstract I in this series) was extended to include 47 rat estrogen receptor (rER) relative binding affinity (RBA) measurements in addition to the...

PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: A COMPARISON OF THREE MODELS

EPA Science Inventory

A comparative analysis of how three COREPA models for ER binding affinity performed when used to predict potential estrogen receptor (ER) ligands is presented. Models I and II were developed based on training sets of 232 and 279 rat ER binding affinity measurements, respectively....

Characterization of nicotine binding to the rat brain P/sub 2/ preparation: the identification of multiple binding sites which include specific up-regulatory site(s)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sloan, J.W.

1984-01-01

These studies show that nicotine binds to the rat brain P/sub 2/ preparation by saturable and reversible processes. Multiple binding sites were revealed by the configuration of saturation, kinetic and Scatchard plots. A least squares best fit of Scatchard data using nonlinear curve fitting programs confirmed the presence of a very high affinity site, an up-regulatory site, a high affinity site and one or two low affinity sites. Stereospecificity was demonstrated for the up-regulatory site where (+)-nicotine was more effective and for the high affinity site where (-)-nicotine had a higher affinity. Drugs which selectively up-regulate nicotine binding site(s) havemore » been identified. Further, separate very high and high affinity sites were identified for (-)- and (+)-(/sup 3/H)nicotine, based on evidence that the site density for the (-)-isomer is 10 times greater than that for the (+)-isomer at these sites. Enhanced nicotine binding has been shown to be a statistically significant phenomenon which appears to be a consequence of drugs binding to specific site(s) which up-regulate binding at other site(s). Although Scatchard and Hill plots indicate positive cooperatively, up-regulation more adequately describes the function of these site(s). A separate up-regulatory site is suggested by the following: (1) Drugs vary markedly in their ability to up-regulate binding. (2) Both the affinity and the degree of up-regulation can be altered by structural changes in ligands. (3) Drugs with specificity for up-regulation have been identified. (4) Some drugs enhance binding in a dose-related manner. (5) Competition studies employing cold (-)- and (+)-nicotine against (-)- and (+)-(/sup 3/H)nicotine show that the isomers bind to separate sites which up-regulate binding at the (-)- and (+)-nicotine high affinity sites and in this regard (+)-nicotine is more specific and efficacious than (-)-nicotine.« less

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turley, E.A.; Moore, D.; Hayden, L.J.

1987-06-02

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weightmore » range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals.

PubMed Central

Bruno, S.; Bettati, S.; Manfredini, M.; Mozzarelli, A.; Bolognesi, M.; Deriu, D.; Rosano, C.; Tsuneshige, A.; Yonetani, T.; Henry, E. R.

2000-01-01

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model. PMID:10794410

Effect of Detergents on Galactoside Binding by Melibiose Permeases.

PubMed

Amin, Anowarul; Hariharan, Parameswaran; Chae, Pil Seok; Guan, Lan

2015-09-29

The effect of various detergents on the stability and function of the melibiose permeases of Escherichia coli (MelBEc) and Salmonella typhimurium (MelBSt) was studied. In n-dodecyl-β-d-maltoside (DDM) or n-undecyl-β-d-maltoside (UDM), WT MelBSt binds melibiose with an affinity similar to that in the membrane. However, with WT MelBEc or MelBSt mutants (Arg141 → Cys, Arg295 → Cys, or Arg363 → Cys), galactoside binding is not detected in these detergents, but binding to the phosphotransferase protein IIA(Glc) is maintained. In the amphiphiles lauryl maltose neopentyl glycol (MNG-3) or glyco-diosgenin (GDN), galactoside binding with all of the MelB proteins is observed, with slightly reduced affinities. MelBSt is more thermostable than MelBEc, and the thermostability of either MelB is largely increased in MNG-3 or GDN. Therefore, the functional defect with DDM or UDM likely results from the relative instability of the sensitive MelB proteins, and stability, as well as galactoside binding, is retained in MNG-3 or GDN. Furthermore, isothermal titration calorimetry of melibiose binding with MelBSt shows that the favorable entropic contribution to the binding free energy is decreased in MNG-3, indicating that the conformational dynamics of MelB is restricted in this detergent.

Effect of detergents on galactoside binding by melibiose permeases

PubMed Central

Amin, Anowarul; Hariharan, Parameswaran; Chae, Pil Seok; Guan, Lan

2015-01-01

The effect of various detergents on the stability and function of melibiose permeases of Escherichia coli (MelBEc) or Salmonella typhimurium (MelBSt) were studied. In n-dodecyl-β-d-maltoside (DDM) or n-undecyl-β-d-maltoside (UDM), WT MelBSt binds melibiose with an affinity similar to that in the membrane. However, with WT MelBEc or MelBSt mutants (Arg141→Cys, Arg295→Cys or Arg363→Cys), galactoside binding is not detected in these detergents, but binding to the phosphotransferase protein IIAGlc is maintained. In the amphiphiles lauryl maltose neopentyl glycol (MNG-3) or glyco-diosgenin (GDN), galactoside binding with all the MelB proteins is observed, with slightly reduced affinities. MelBSt is more thermostable than MelBEc, and the thermostability of either MelB is largely increased in MNG-3 or GDN. Therefore, the functional defect with DDM or UDM likely results from relative instability of the sensitive MelB proteins, and stability, as well as galactoside binding, is retained in MNG-3 or GDN. Furthermore, isothermal titration calorimetry of melibiose binding with MelBSt shows that the favorable entropic contribution to the binding free energy is decreased in MNG-3, indicating that the conformational dynamics of MelB is restricted in this detergent. PMID:26352464

Ion-binding properties of Calnuc, Ca2+ versus Mg2+--Calnuc adopts additional and unusual Ca2+-binding sites upon interaction with G-protein.

PubMed

Kanuru, Madhavi; Samuel, Jebakumar J; Balivada, Lavanya M; Aradhyam, Gopala K

2009-05-01

Calnuc is a novel, highly modular, EF-hand containing, Ca(2+)-binding, Golgi resident protein whose functions are not clear. Using amino acid sequences, we demonstrate that Calnuc is a highly conserved protein among various organisms, from Ciona intestinalis to humans. Maximum homology among all sequences is found in the region that binds to G-proteins. In humans, it is known to be expressed in a variety of tissues, and it interacts with several important protein partners. Among other proteins, Calnuc is known to interact with heterotrimeric G-proteins, specifically with the alpha-subunit. Herein, we report the structural implications of Ca(2+) and Mg(2+) binding, and illustrate that Calnuc functions as a downstream effector for G-protein alpha-subunit. Our results show that Ca(2+) binds with an affinity of 7 mum and causes structural changes. Although Mg(2+) binds to Calnuc with very weak affinity, the structural changes that it causes are further enhanced by Ca(2+) binding. Furthermore, isothermal titration calorimetry results show that Calnuc and the G-protein bind with an affinity of 13 nm. We also predict a probable function for Calnuc, that of maintaining Ca(2+) homeostasis in the cell. Using Stains-all and terbium as Ca(2+) mimic probes, we demonstrate that the Ca(2+)-binding ability of Calnuc is governed by the activity-based conformational state of the G-protein. We propose that Calnuc adopts structural sites similar to the ones seen in proteins such as annexins, c2 domains or chromogrannin A, and therefore binds more calcium ions upon binding to Gialpha. With the number of organelle-targeted G-protein-coupled receptors increasing, intracellular communication mediated by G-proteins could become a new paradigm. In this regard, we propose that Calnuc could be involved in the downstream signaling of G-proteins.

The histidine phosphocarrier protein, HPr, binds to the highly thermostable regulator of sigma D protein, Rsd, and its isolated helical fragments.

PubMed

Neira, José L; Hornos, Felipe; Cozza, Concetta; Cámara-Artigas, Ana; Abián, Olga; Velázquez-Campoy, Adrián

2018-02-01

The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ 70 factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPr sc , and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol -1 at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPr sc was 2 μM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPr sc in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPr sc , HPr 9-30 , comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPr sc was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPr sc . This implies that specific fragments of Rsd and HPr sc can be used to interfere with other protein-protein interactions (PPIs) of each other protein. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

PubMed

Ciossek, T; Ullrich, A

1997-01-09

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation

PubMed Central

Zhao, Bin-Bin; Gong, Lu-Lu; Jin, Wen-Jing; Liu, Jing-Jun; Wang, Jing-Fei; Wang, Tian-Tian; Yuan, Xiao-Hui; He, You-Wen

2013-01-01

1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization. PMID:24278299

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Binding affinity of aluminium to human serum transferrin and effects of carbohydrate chain modification as studied by HPLC/high-resolution ICP-MS--speciation of aluminium in human serum.

PubMed

Nagaoka, Megumi Hamano; Maitani, Tamio

2005-09-01

Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented.

Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

PubMed

Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

2015-12-01

Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides.

PubMed

Zhang, Pengzhi; Tripathi, Swarnendu; Trinh, Hoa; Cheung, Margaret S

2017-03-28

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca 2+ ) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca 2+ /CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca 2+ signaling pathways in neurons. It was shown experimentally that Ca 2+ /CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca 2+ -free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca 2+ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng 13-49 ), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng 13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng 13-49 structures by matching the changes in the chemical shifts of CaM upon Ng 13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca 2+ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca 2+ for CaM in the presence of Ng 13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca 2+ binding loops particularly at C-domain of CaM, enabling Ca 2+ release. In contrast, CaMKII peptide increases Ca 2+ affinity for the C-domain of CaM by stabilizing the two Ca 2+ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng 13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca 2+ binding affinities. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Bioinorganic Chemistry of Parkinson's Disease: Affinity and Structural Features of Cu(I) Binding to the Full-Length β-Synuclein Protein.

PubMed

Miotto, Marco C; Pavese, Mayra D; Quintanar, Liliana; Zweckstetter, Markus; Griesinger, Christian; Fernández, Claudio O

2017-09-05

Alterations in the levels of copper in brain tissue and formation of α-synuclein (αS)-copper complexes might play a key role in the amyloid aggregation of αS and the onset of Parkinson's disease (PD). Recently, we demonstrated that formation of the high-affinity Cu(I) complex with the N-terminally acetylated form of the protein αS substantially increases and stabilizes local conformations with α-helical secondary structure and restricted motility. In this work, we performed a detailed NMR-based structural characterization of the Cu(I) complexes with the full-length acetylated form of its homologue β-synuclein (βS), which is colocalized with αS in vivo and can bind copper ions. Our results show that, similarly to αS, the N-terminal region of βS constitutes the preferential binding interface for Cu(I) ions, encompassing two independent and noninteractive Cu(I) binding sites. According to these results, βS binds the metal ion with higher affinity than αS, in a coordination environment that involves the participation of Met-1, Met-5, and Met-10 residues (site 1). Compared to αS, the shift of His from position 50 to 65 in the N-terminal region of βS does not change the Cu(I) affinity features at that site (site 2). Interestingly, the formation of the high-affinity βS-Cu(I) complex at site 1 in the N-terminus promotes a short α-helix conformation that is restricted to the 1-5 segment of the AcβS sequence, which differs with the substantial increase in α-helix conformations seen for N-terminally acetylated αS upon Cu(I) complexation. Our NMR data demonstrate conclusively that the differences observed in the conformational transitions triggered by Cu(I) binding to AcαS and AcβS find a correlation at the level of their backbone dynamic properties; added to the potential biological implications of these findings, this fact opens new avenues of investigations into the bioinorganic chemistry of PD.

Modulation of DNA binding by gene-specific transcription factors.

PubMed

Schleif, Robert F

2013-10-01

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

PubMed

Root-Bernstein, Robert; Turke, Miah; Subhramanyam, Udaya K Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-17

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

Production and Characterization of Desmalonichrome Relative Binding Affinity for Uranyl Ions in Relation to Other Siderophores

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mo, Kai-For; Dai, Ziyu; Wunschel, David S.

2016-06-24

Siderophores are Fe binding secondary metabolites that have been investigated for their uranium binding properties. Much of the previous work has focused on characterizing hydroxamate types of siderophores, such as desferrioxamine B, for their uranyl binding affinity. Carboxylate forms of these metabolites hold potential to be more efficient chelators of uranyl, yet they have not been widely studied and are more difficult to obtain. Desmalonichrome is a carboxylate siderophore which is not commercially available and so was obtained from the ascomycete fungus Fusarium oxysporum cultivated under Fe depleted conditions. The relative affinity for uranyl binding of desmalonichrome was investigated usingmore » a competitive analysis of binding affinities between uranyl acetate and different concentrations of iron(III) chloride using electrospray ionization mass spectrometry (ESI-MS). In addition to desmalonichrome, three other siderophores, including two hydroxamates (desferrioxamine B and desferrichrome) and one carboxylate (desferrichrome A) were studied to understand their relative affinities for the uranyl ion at two pH values. The binding affinities of hydroxymate siderophores to uranyl ion were found to decrease to a greater degree at lower pH as the concentration of Fe (III) ion increases. On the other hand, lowering pH has little impact on the binding affinities between carboxylate siderophores and uranyl ion. Desmalonichrome was shown to have the greatest relative affinity for uranyl at any pH and Fe(III) concentration. These results suggest that acidic functional groups in the ligands are critical for strong chelation with uranium at lower pH.« less

Caffeine and Sugars Interact in Aqueous Solutions: A Simulation and NMR Study

PubMed Central

Tavagnacco, Letizia; Engström, Olof; Schnupf, Udo; Saboungi, Marie-Louise; Himmel, Michael; Widmalm, Göran; Cesàro, Attilio; Brady, John W.

2012-01-01

Molecular dynamics simulations were carried out on several systems of caffeine interacting with simple sugars. These included a single caffeine molecule in a 3 molal solution of α-D-glucopyranose, at a caffeine concentration of 0.083 molal; a single caffeine in a 3 molal solution of β-D-glucopyranose, and a single caffeine molecule in a 1.08 molal solution of sucrose (table sugar). Parallel Nuclear Magnetic Resonance titration experiments were carried out on the same solutions under similar conditions. Consistent with previous thermodynamic experiments, the sugars were found to have an affinity for the caffeine molecules in both the simulations and experiments, and that the binding in these complexes occurs by face-to-face stacking of the hydrophobic triad of protons of the pyranose rings against the caffeine face, rather than by hydrogen bonding. For the disaccharide, the binding occurs via stacking of the glucose ring against the caffeine, with a lesser affinity for the fructose observed. These findings are consistent with the association being driven by hydrophobic hydration, and are similar to the previously observed binding of glucose rings to various other planar molecules, including indole, serotonin, and phenol. PMID:22897449

Caffeine and sugars interact in aqueous solutions: a simulation and NMR study.

PubMed

Tavagnacco, Letizia; Engström, Olof; Schnupf, Udo; Saboungi, Marie-Louise; Himmel, Michael; Widmalm, Göran; Cesàro, Attilio; Brady, John W

2012-09-27

Molecular dynamics simulations were carried out on several systems of caffeine interacting with simple sugars. These included a single caffeine molecule in a 3 m solution of α-D-glucopyranose, at a caffeine concentration of 0.083 m, a single caffeine in a 3 m solution of β-D-glucopyranose, and a single caffeine molecule in a 1.08 m solution of sucrose (table sugar). Parallel nuclear magnetic resonance titration experiments were carried out on the same solutions under similar conditions. Consistent with previous thermodynamic experiments, the sugars were found to have an affinity for the caffeine molecules in both the simulations and experiments, and the binding in these complexes occurs by face-to-face stacking of the hydrophobic triad of protons of the pyranose rings against the caffeine face, rather than by hydrogen bonding. For the disaccharide, the binding occurs via stacking of the glucose ring against the caffeine, with a lesser affinity for the fructose observed. These findings are consistent with the association being driven by hydrophobic hydration and are similar to the previously observed binding of glucose rings to various other planar molecules, including indole, serotonin, and phenol.

Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlstedt-Duke, J.; Stroemstedt, P.E.; Persson, B.

1988-05-15

Purified rat liver glucocorticoid receptor was covalently charged with (/sup 3/H)glucocorticoid by photoaffinity labeling (UV irradiation of (/sup 3/H)triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with (/sup 3/H)dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with (/sup 3/H)triamcinolone acetonide and Cys-656 after affinity labeling with (/sup 3/H)dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. Themore » patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.« less

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

PubMed

Mithöfer, A; Fliegmann, J; Neuhaus-Url, G; Schwarz, H; Ebel, J

2000-08-01

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Characterization of [3H] oxymorphone binding sites in mouse brain: Quantitative autoradiography in opioid receptor knockout mice.

PubMed

Yoo, Ji Hoon; Borsodi, Anna; Tóth, Géza; Benyhe, Sándor; Gaspar, Robert; Matifas, Audrey; Kieffer, Brigitte L; Metaxas, Athanasios; Kitchen, Ian; Bailey, Alexis

2017-03-16

Oxymorphone, one of oxycodone's metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear. This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [ 3 H]oxymorphone revealed high affinity binding sites in mouse brain displaying Kd of 1.7nM and Bmax of 147fmol/mg. Furthermore, we performed quantitative autoradiography binding studies using [ 3 H]oxymorphone in mouse brain. The distribution of [ 3 H]oxymorphone binding sites was found to be similar to the selective MOP agonist [ 3 H]DAMGO in the mouse brain. [ 3 H]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [ 3 H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP, and not the DOP or the KOP is the main high affinity binding target for oxymorphone. Copyright © 2017 Elsevier B.V. All rights reserved.

Design and synthesis of N-(3,3-diphenylpropenyl)alkanamides as a novel class of high-affinity MT2-selective melatonin receptor ligands.

PubMed

Bedini, Annalida; Spadoni, Gilberto; Gatti, Giuseppe; Lucarini, Simone; Tarzia, Giorgio; Rivara, Silvia; Lorenzi, Simone; Lodola, Alessio; Mor, Marco; Lucini, Valeria; Pannacci, Marilou; Scaglione, Francesco

2006-12-14

A novel series of melatonin receptor ligands was discovered by opening the cyclic scaffolds of known classes of high affinity melatonin receptor antagonists, while retaining the pharmacophore elements postulated by previously described 3D-QSAR and receptor models. Compounds belonging to the classes of 2,3- and [3,3-diphenylprop(en)yl]alkanamides and of o- or [(m-benzyl)phenyl]ethyl-alkanamides were synthesized and tested on MT(1) and MT(2) receptors. The class of 3,3-diphenyl-propenyl-alkanamides was the most interesting one, with compounds having MT(2) receptor affinity similar to that of MLT, remarkable MT(2) selectivity, and partial agonist or antagonist behavior. In particular, the (E)-m-methoxy cyclobutanecarboxamido derivative 18f and the di-(m-methoxy) acetamido one, 18g, have sub-nM affinity for the MT(2) subtype, with more than 100-fold selectivity over MT(1), 18f being an antagonist and 18g a partial agonist on GTPgammaS test. Docking of 18g into a previously developed MT(2) receptor model showed a binding scheme consistent with that of other antagonists. The MT(2) expected binding affinities of the new compounds were calculated by a previously developed 3D-QSAR CoMFA model, giving satisfactory predictions.

Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Lianying; College of Life Science, Dezhou University, Dezhou 253023; Ren, Xiao-Min

2014-09-15

Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group.more » For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.« less

Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

PubMed

Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

2014-07-15

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

Specificity and Affinity Quantification of Flexible Recognition from Underlying Energy Landscape Topography

PubMed Central

Chu, Xiakun; Wang, Jin

2014-01-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition. PMID:25144525

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Specificity and affinity quantification of flexible recognition from underlying energy landscape topography.

PubMed

Chu, Xiakun; Wang, Jin

2014-08-01

Flexibility in biomolecular recognition is essential and critical for many cellular activities. Flexible recognition often leads to moderate affinity but high specificity, in contradiction with the conventional wisdom that high affinity and high specificity are coupled. Furthermore, quantitative understanding of the role of flexibility in biomolecular recognition is still challenging. Here, we meet the challenge by quantifying the intrinsic biomolecular recognition energy landscapes with and without flexibility through the underlying density of states. We quantified the thermodynamic intrinsic specificity by the topography of the intrinsic binding energy landscape and the kinetic specificity by association rate. We found that the thermodynamic and kinetic specificity are strongly correlated. Furthermore, we found that flexibility decreases binding affinity on one hand, but increases binding specificity on the other hand, and the decreasing or increasing proportion of affinity and specificity are strongly correlated with the degree of flexibility. This shows more (less) flexibility leads to weaker (stronger) coupling between affinity and specificity. Our work provides a theoretical foundation and quantitative explanation of the previous qualitative studies on the relationship among flexibility, affinity and specificity. In addition, we found that the folding energy landscapes are more funneled with binding, indicating that binding helps folding during the recognition. Finally, we demonstrated that the whole binding-folding energy landscapes can be integrated by the rigid binding and isolated folding energy landscapes under weak flexibility. Our results provide a novel way to quantify the affinity and specificity in flexible biomolecular recognition.

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

PubMed

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.

PubMed

Lan, Hongxiang; Liu, Yong; Bell, Michal I; Gurevich, Vsevolod V; Neve, Kim A

2009-01-01

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.

How Much Binding Affinity Can be Gained by Filling a Cavity?

PubMed Central

Kawasaki, Yuko; Chufan, Eduardo E.; Lafont, Virginie; Hidaka, Koushi; Kiso, Yoshiaki; Amzel, L. Mario; Freire, Ernesto

2011-01-01

Binding affinity optimization is critical during drug development. Here we evaluate the thermodynamic consequences of filling a binding cavity with functionalities of increasing van der Waals radii (-H, -F, -Cl and CH3) that improve the geometric fit without participating in hydrogen bonding or other specific interactions. We observe a binding affinity increase of two orders of magnitude. There appears to be three phases in the process. The first phase is associated with the formation of stable van der Waals interactions. This phase is characterized by a gain in binding enthalpy and a loss in binding entropy, attributed to a loss of conformational degrees of freedom. For the specific case presented in this paper, the enthalpy gain amounts to −1.5 kcal/mol while the entropic losses amount to +0.9 kcal/mol resulting in a net 3.5-fold affinity gain. The second phase is characterized by simultaneous enthalpic and entropic gains. This phase improves the binding affinity 25-fold. The third phase represents the collapse of the trend and is triggered by the introduction of chemical functionalities larger than the binding cavity itself (CH(CH3)2). It is characterized by large enthalpy and affinity losses. The thermodynamic signatures associated with each phase provide guidelines for lead optimization. PMID:20028396

Specificity of cell–cell adhesion by classical cadherins: Critical role for low-affinity dimerization through β-strand swapping

PubMed Central

Chen, Chien Peter; Posy, Shoshana; Ben-Shaul, Avinoam; Shapiro, Lawrence; Honig, Barry H.

2005-01-01

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell–cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by “swapping” the N-terminal β-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that “closed” monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell–cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems. PMID:15937105

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which oftenmore » takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.« less

Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography-mass spectrometry for solution-based ligand screening against multiple proteins.

PubMed

Zhou, Jian-Liang; An, Jing-Jing; Li, Ping; Li, Hui-Jun; Jiang, Yan; Cheng, Jie-Fei

2009-03-20

We present herein a novel bioseparation/chemical analysis strategy for protein-ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography-mass spectrometry (LC-MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC-MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5s, and on-line prepare the "clean" sample to be directly compatible with the LC-MS analysis. The improvement in performance of this 2D-TFC/LC-MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC-MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Sun; Li, Shu-Xing; Bren, Nina

2013-09-01

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Å 2 (1 Å=0.1 nm) of surface area, within which Arg 36 and Phe 32 from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr 184 from loop-C of α7, while Asp 30 of α-btx forms a hydrogen bond with the hydroxy group of Tyr 184. These inter-residue interactions diverge from thosemore » in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr 184 with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.« less

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

Role of 'B-b' knob-hole interactions in fibrin binding to adsorbed fibrinogen.

PubMed

Geer, C B; Tripathy, A; Schoenfisch, M H; Lord, S T; Gorkun, O V

2007-12-01

The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs 'A' and 'B' exposed by thrombin cleavage and polymerization holes 'a' and 'b' present in fibrinogen and fibrin. Although structural studies have defined the 'A-a' and 'B-b' interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin. We designed experiments to determine the affinities of knob-hole interactions, either 'A-a' alone or 'A-a' and 'B-b' together. We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing 'A' knobs, desA-NDSK, or both 'A' and 'B' knobs, desAB-NDSK. The desA- and desAB-NDSK fragments bound to fibrinogen with statistically similar K(d)'s of 5.8 +/- 1.1 microm and 3.7 +/- 0.7 microm (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic 'A' knob peptide GPRP and synthetic 'B' knob peptides GHRP and AHRPY, inhibited the binding of desA- and/or desAB-NDSK. The peptide inhibition findings show both 'A-a' and 'B-b' interactions participate in desAB-NDSK binding to fibrinogen, indicating 'B-b' interactions can occur simultaneously with 'A-a'. Furthermore, 'A-a' interactions are much stronger than 'B-b' because the affinity of desA-NDSK was not markedly different from desAB-NDSK.

Batrachotoxin Changes the Properties of the Muscarinic Receptor in Rat Brain and Heart: Possible Interaction(s) between Muscarinic Receptors and Sodium Channels

NASA Astrophysics Data System (ADS)

Cohen-Armon, Malca; Kloog, Yoel; Henis, Yoav I.; Sokolovsky, Mordechai

1985-05-01

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Earle, M.; Concas, A.; Yamamura, H.I.

1986-03-01

The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. atmore » 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.« less

Free-energy simulations reveal that both hydrophobic and polar interactions are important for influenza hemagglutinin antibody binding.

PubMed

Xia, Zhen; Huynh, Tien; Kang, Seung-gu; Zhou, Ruhong

2012-03-21

Antibodies binding to conserved epitopes can provide a broad range of neutralization to existing influenza subtypes and may also prevent the propagation of potential pandemic viruses by fighting against emerging strands. Here we propose a computational framework to study structural binding patterns and detailed molecular mechanisms of viral surface glycoprotein hemagglutinin (HA) binding with a broad spectrum of neutralizing monoclonal antibody fragments (Fab). We used rigorous free-energy perturbation (FEP) methods to calculate the antigen-antibody binding affinities, with an aggregate underlying molecular-dynamics simulation time of several microseconds (∼2 μs) using all-atom, explicit-solvent models. We achieved a high accuracy in the validation of our FEP protocol against a series of known binding affinities for this complex system, with <0.5 kcal/mol errors on average. We then introduced what to our knowledge are novel mutations into the interfacial region to further study the binding mechanism. We found that the stacking interaction between Trp-21 in HA2 and Phe-55 in the CDR-H2 of Fab is crucial to the antibody-antigen association. A single mutation of either W21A or F55A can cause a binding affinity decrease of ΔΔG > 4.0 kcal/mol (equivalent to an ∼1000-fold increase in the dissociation constant K(d)). Moreover, for group 1 HA subtypes (which include both the H1N1 swine flu and the H5N1 bird flu), the relative binding affinities change only slightly (< ±1 kcal/mol) when nonpolar residues at the αA helix of HA mutate to conservative amino acids of similar size, which explains the broad neutralization capability of antibodies such as F10 and CR6261. Finally, we found that the hydrogen-bonding network between His-38 (in HA1) and Ser-30/Gln-64 (in Fab) is important for preserving the strong binding of Fab against group 1 HAs, whereas the lack of such hydrogen bonds with Asn-38 in most group 2 HAs may be responsible for the escape of antibody neutralization. These large-scale simulations may provide new insight into the antigen-antibody binding mechanism at the atomic level, which could be essential for designing more-effective vaccines for influenza. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Evaluation of 111In-labeled EPep and FibPep as tracers for fibrin SPECT imaging.

PubMed

Starmans, Lucas W E; van Duijnhoven, Sander M J; Rossin, Raffaella; Berben, Monique; Aime, Silvio; Daemen, Mat J A P; Nicolay, Klaas; Grüll, Holger

2013-11-04

Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an (111)In-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel (111)In-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging. In vitro, both EPep and FibPep showed high stability in serum, but were less stable in liver and kidney homogenate assays. Both peptide probes displayed comparable affinities toward human and mouse derived fibrin (Kd ≈ 1 μM), and similarly to FibPep, EPep showed fast blood clearance, low nontarget uptake and high thrombus uptake (6.8 ± 1.2% ID g(-1)) in a mouse carotid artery thrombosis model. Furthermore, EPep showed a similar affinity toward rat derived fibrin (Kd ≈ 1 μM), displayed high thrombus uptake in a rat carotid artery thrombosis model (0.74 ± 0.39% ID g(-1)), and allowed sensitive detection of thrombosis in rats using SPECT. In contrast, FibPep displayed a significantly lower affinity toward rat derived fibrin (Kd ≈ 14 μM) and low uptake in rat thrombi (0.06 ± 0.02% ID g(-1)) and did not allow clear visualization of carotid artery thrombosis in rats using SPECT. These results were confirmed ex vivo by autoradiography, which showed a 7-fold higher ratio of activity in the thrombus over the contralateral carotid artery for EPep in comparison to FibPep. These findings suggest that the FibPep binding fibrin epitope is not fully homologous between humans and rats, and that preclinical rat models of disease should not be employed to gauge the clinical potential of FibPep. In conclusion, both peptides showed approximately similar metabolic stability and affinity toward human and mouse derived fibrin, and displayed high thrombus uptake in a mouse carotid artery thrombosis model. Therefore, both EPep and FibPep are promising fibrin targeted tracers for translation into clinical settings to serve as novel tools for molecular imaging of fibrin.

Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

PubMed

Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

2014-07-18

This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of glycation on meloxicam binding to human serum albumin

NASA Astrophysics Data System (ADS)

Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

2011-05-01

The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein.

PubMed

Gillard, Michel; Chatelain, Pierre; Fuks, Bruno

2006-04-24

A specific binding site for the antiepileptic drug levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra) in rat brain, referred to as the levetiracetam binding site, was discovered several years ago. More recently, this binding site has been identified as the synaptic vesicle protein 2A (SV2A), a protein present in synaptic vesicles [Lynch, B., Lambeng, N., Nocka, K., Kensel-Hammes, P., Bajjalieh, S.M., Matagne, A., Fuks, B., 2004. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc. Natl. Acad. Sci. USA, 101, 9861-9866.]. In this study, we characterized the binding properties of levetiracetam in post-mortem human brain and compared them to human SV2A expressed in Chinese hamster ovary (CHO) cells. The results showed that the binding properties of levetiracetam and [3H]ucb 30889, an analogue that was previously characterized as a suitable ligand for levetiracetam binding site/SV2A in rat brain [Gillard, M., Fuks, B., Michel, P., Vertongen, P., Massingham, R. Chatelain, P., 2003. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur. J. Pharmacol. 478, 1-9.], are almost identical in human brain samples (cerebral cortex, hippocampus and cerebellum) and in CHO cell membranes expressing the human SV2A protein. Moreover, the results are also similar to those previously obtained in rat brain. [3H]ucb 30889 binding in human brain and to SV2A was saturable and reversible. At 4 degrees C, its binding kinetics were best fitted assuming a two-phase model in all tissues. The half-times of association for the fast component ranged between 1 to 2 min and represent 30% to 36% of the sites whereas the half-times for the slow component ranged from 20 to 29 min. In dissociation experiments, the half-times were from 2 to 4 min for the fast component (33% to 49% of the sites) and 20 to 41 min for the slow component. Saturation binding curves led to Kd values for [3H]ucb 30889 of 53+/-7, 55+/-9, 70+/-11 and 75+/-33 nM in human cerebral cortex, hippocampus, cerebellum and CHO cells expressing SV2A respectively. Bmax values around 3-4 pmol/mg protein were calculated in all brain regions. Some of the saturation curves displayed curvilinear Scatchard plots indicating the presence of high and low affinity binding sites. When this was the case, Kd values from 25 to 30 nM for the high affinity sites (24% to 34% of total sites) and from 200 to 275 nM for the low affinity sites were calculated. This was observed in all brain regions and in CHO cell membranes expressing the SV2A protein. It cannot be explained by putative binding of [3H]ucb 30889 to SV2B or C isoforms but may reflect different patterns of SV2A glycosylation or the formation of SV2A oligomers. Competition experiments were performed to determine the affinities for SV2A of a variety of compounds including levetiracetam, some of its analogues and other molecules known to interact with levetiracetam binding sites in rat brain such as bemegride, pentylenetetrazol and chlordiazepoxide. We found an excellent correlation between the affinities of these compounds measured in human brain, rat brain and CHO cells expressing human SV2A. In conclusion, we report for the first time that the binding characteristics of native levetiracetam binding sites/SV2A in human brain and rat brain share very similar properties with human recombinant SV2A expressed in CHO cells.

Purification of L-( sup 3 H) Nicotine eliminates low affinity binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romm, E.; Marks, M.J.; Collins, A.C.

1990-01-01

Some studies of L-({sup 3}H) nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicated that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-({sup 3}H)nicotine eliminates the low affinity site.

Differential mechanisms of binding of anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA to E. coli RNA polymerase lead to diverse physiological consequences.

PubMed

Sharma, Umender K; Chatterji, Dipankar

2008-05-01

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to sigma(70) with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences▿

PubMed Central

Sharma, Umender K.; Chatterji, Dipankar

2008-01-01

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70. PMID:18359804

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.

PubMed

Raffaï, R; Weisgraber, K H; MacKenzie, R; Rupp, B; Rassart, E; Hirama, T; Innerarity, T L; Milne, R

2000-03-10

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.

PubMed Central

Dubois, B W; Cherian, S F; Evers, A S

1993-01-01

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein. PMID:8341659

The FOXP2 forkhead domain binds to a variety of DNA sequences with different rates and affinities.

PubMed

Webb, Helen; Steeb, Olga; Blane, Ashleigh; Rotherham, Lia; Aron, Shaun; Machanick, Philip; Dirr, Heini; Fanucchi, Sylvia

2017-07-01

FOXP2 is a member of the P subfamily of FOX transcription factors, the DNA-binding domain of which is the winged helix forkhead domain (FHD). In this work we show that the FOXP2 FHD is able to bind to various DNA sequences, including a novel sequence identified in this work, with different affinities and rates as detected using surface plasmon resonance. Combining the experimental work with molecular docking, we show that high-affinity sequences remain bound to the protein for longer, form a greater number of interactions with the protein and induce a greater structural change in the protein than low-affinity sequences. We propose a binding model for the FOXP2 FHD that involves three types of binding sequence: low affinity sites which allow for rapid scanning of the genome by the protein in a partially unstructured state; moderate affinity sites which serve to locate the protein near target sites and high-affinity sites which secure the protein to the DNA and induce a conformational change necessary for functional binding and the possible initiation of downstream transcriptional events. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Engineering high-potency R-spondin adult stem cell growth factors.

PubMed

Warner, Margaret L; Bell, Tufica; Pioszak, Augen A

2015-01-01

Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 > RSPO2/3 > RSPO1 and ZNRF3 with affinity rank order RSPO2/3 > > RSPO1 > RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1 LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands.

PubMed Central

Kinnally, K W; Zorov, D B; Antonenko, Y N; Snyder, S H; McEnery, M W; Tedeschi, H

1993-01-01

The mitochrondrial benzodiazepine receptor (mBzR) binds a subset of benzodiazepines and isoquinoline carboxamides with nanomolar affinity and consists of the voltage-dependent anion channel, the adenine nucleotide translocator, and an 18-kDa protein. The effect of ligands of the mBzR on two inner mitochondrial membrane channel activities was determined with patch-clamp techniques. The relative inhibitory potencies of the drugs resemble their binding affinities for the mBzR. Ro5-4864 and protoporphyrin IX inhibit activity of the multiple conductance channel (MCC) and the mitochondrial centum-picosiemen (mCtS) channel activities at nanomolar concentrations. PK11195 inhibits mCtS activity at similar levels. Higher concentrations of protoporphyrin IX induce MCC but possibly not mCtS activity. Clonazepam, which has low affinity for mBzR, is at least 500 times less potent at both channel activities. Ro15-1788, which also has a low mBzR affinity, inhibits MCC at very high concentrations (16 microM). The findings indicate an association of these two channel activities with the proteins forming the mBzR complex and are consistent with an interaction of inner and outer membrane channels. PMID:7679505

DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

PubMed Central

Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

2009-01-01

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

Reproducing Crystal Binding Modes of Ligand Functional Groups using Site-Identification by Ligand Competitive Saturation (SILCS) Simulations

PubMed Central

Raman, E. Prabhu; Yu, Wenbo; Guvench, Olgun; MacKerell, Alexander D.

2011-01-01

The applicability of a computational method, Site Identification by Ligand Competitive Saturation (SILCS), to identify regions on a protein surface with which different types of functional groups on low-molecular weight inhibitors interact is demonstrated. The method involves molecular dynamics (MD) simulations of a protein in an aqueous solution of chemically diverse small molecules from which probability distributions of fragments types, termed FragMaps, are obtained. In the present application, SILCS simulations are performed with an aqueous solution of 1 M benzene and propane to map the affinity pattern of the protein for aromatic and aliphatic functional groups. In addition, water hydrogen and oxygen atoms serve as probes for hydrogen bond donor and acceptor affinity, respectively. The method is tested using a set of 7 proteins for which crystal structures of complexes with several high affinity inhibitors are known. Good agreement is obtained between FragMaps and the positions of chemically similar functional groups in inhibitors as observed in the X-ray crystallographic structures. Quantitative capabilities of the SILCS approach are demonstrated by converting FragMaps to free energies, termed Grid Free Energies (GFE), and showing correlation between the GFE values and experimental binding affinities. For proteins for which ligand decoy sets are available, GFE values are shown to typically score the crystal conformation and conformations similar to it more favorable than decoys. Additionally, SILCS is tested for its ability to capture the subtle differences in ligand affinity across homologous proteins, information which may be of utility towards specificity-guided drug design. Taken together, our results show that SILCS can recapitulate the known location of functional groups of bound inhibitors for a number of proteins, suggesting that the method may be of utility for rational drug design. PMID:21456594

Responses of normal and sickle cell hemoglobin to S-nitroscysteine: implications for therapeutic applications of NO in treatment of sickle cell disease.

PubMed

Bonaventura, Celia; Godette, Gerald; Ferruzzi, Giulia; Tesh, Shirley; Stevens, Robert D; Henkens, Robert

2002-07-10

Factors which govern transnitrosation reactions between hemoglobin (Hb) and low molecular weight thiols may define the extent to which S-nitrosated Hb (SNO-Hb) plays a role in NO in the control of blood pressure and other NO-dependent reactions. We show that exposure to S-nitrosylated cysteine (CysNO) produces equivalent levels of SNO-Hb for Hb A(0) and sickle cell Hb (Hb S), although these proteins differ significantly in the electron affinity of their heme groups as measured by their anaerobic redox potentials. Dolphin Hb, a cooperative Hb with a redox potential like that of Hb S, produces less SNO-Hb, indicating that steric considerations outweigh effects of altered electron affinity at the active-site heme groups in control of SNO-Hb formation. Examination of oxygen binding at 5-20 mM heme concentrations revealed increases due to S-nitrosation in the apparent oxygen affinity of both Hb A(0) and Hb S, similar to increases seen at lower heme concentrations. As observed at lower heme levels, deoxygenation is not sufficient to trigger release of NO from SNO-Hb. A sharp increase in apparent oxygen affinity occurs for unmodified Hb S at concentrations above 12.5 mM, its minimum gelling concentration. This affinity increase still occurs in 30 and 60% S-nitrosated samples, but at higher heme concentration. This oxygen binding behavior is accompanied by decreased gel formation of the deoxygenated protein. S-nitrosation is thus shown to have an effect similar to that reported for other SH-group modifications of Hb S, in which R-state stabilization opposes Hb S aggregation.

Template CoMFA Generates Single 3D-QSAR Models that, for Twelve of Twelve Biological Targets, Predict All ChEMBL-Tabulated Affinities

PubMed Central

Cramer, Richard D.

2015-01-01

The possible applicability of the new template CoMFA methodology to the prediction of unknown biological affinities was explored. For twelve selected targets, all ChEMBL binding affinities were used as training and/or prediction sets, making these 3D-QSAR models the most structurally diverse and among the largest ever. For six of the targets, X-ray crystallographic structures provided the aligned templates required as input (BACE, cdk1, chk2, carbonic anhydrase-II, factor Xa, PTP1B). For all targets including the other six (hERG, cyp3A4 binding, endocrine receptor, COX2, D2, and GABAa), six modeling protocols applied to only three familiar ligands provided six alternate sets of aligned templates. The statistical qualities of the six or seven models thus resulting for each individual target were remarkably similar. Also, perhaps unexpectedly, the standard deviations of the errors of cross-validation predictions accompanying model derivations were indistinguishable from the standard deviations of the errors of truly prospective predictions. These standard deviations of prediction ranged from 0.70 to 1.14 log units and averaged 0.89 (8x in concentration units) over the twelve targets, representing an average reduction of almost 50% in uncertainty, compared to the null hypothesis of “predicting” an unknown affinity to be the average of known affinities. These errors of prediction are similar to those from Tanimoto coefficients of fragment occurrence frequencies, the predominant approach to side effect prediction, which template CoMFA can augment by identifying additional active structural classes, by improving Tanimoto-only predictions, by yielding quantitative predictions of potency, and by providing interpretable guidance for avoiding or enhancing any specific target response. PMID:26065424

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography.

PubMed

Tao, Pingyang; Poddar, Saumen; Sun, Zuchen; Hage, David S; Chen, Jianzhong

2018-02-02

Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets. Copyright © 2018 Elsevier Inc. All rights reserved.

Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poat, J.A.; Cripps, H.E.; Iversen, L.L.

1988-05-01

Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no furthermore » increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.« less

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

PubMed

Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins.

PubMed

Hewitt, Sarah H; Filby, Maria H; Hayes, Ed; Kuhn, Lars T; Kalverda, Arnout P; Webb, Michael E; Wilson, Andrew J

2017-01-17

Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cyt c. High-field natural abundance 1 H, 15 N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photo-Activated Localization Microscopy of Single Carbohydrate Binding Modules on Cellulose Nanofibers

NASA Astrophysics Data System (ADS)

Hor, Amy; Dagel, Daryl; Luu, Quocanh; Savaikar, Madhusudan; Ding, Shi-You; Smith, Steve

2015-03-01

Photo Activated Localization Microscopy (PALM) is used to conduct an in vivo study of the binding affinity of polysaccharide-specific Carbohydrate Binding Modules (CBMs) to insoluble cellulose substrates. Two families of CBMs, namely TrCBM1 and CtCBM3, were modified to incorporate photo-activatable mCherry fluorescent protein (PAmCherry), and exposed to highly crystalline Valonia cellulose nano-fibrils. The resulting PALM images show CBMs binding along the nano-fibril long axis in a punctuated linear array, localized with, on average, 10 nm precision. Statistical analysis of the binding events results in nearest neighbor distributions between CBMs. A comparison between TrCBM1 and CtCBM3 reveals a similarity in the nearest neighbor distribution peaks but differences in the overall binding density. The former is attributed to steric hindrance among the CBMs on the nano-fibril whereas the latter is attributed to differences in the CBMs' binding strength. These results are compared to similar distributions derived from TEM measurements of dried samples of CtCBM3-CdSs quantum dot bioconjugates and AFM images of CtCBM3-GFP bound to similar Valonia nano-fibrils. Funding provided by NSF MPS/DMR/BMAT Award # 1206908.

Calculation of Host-Guest Binding Affinities Using a Quantum-Mechanical Energy Model.

PubMed

Muddana, Hari S; Gilson, Michael K

2012-06-12

The prediction of protein-ligand binding affinities is of central interest in computer-aided drug discovery, but it is still difficult to achieve a high degree of accuracy. Recent studies suggesting that available force fields may be a key source of error motivate the present study, which reports the first mining minima (M2) binding affinity calculations based on a quantum mechanical energy model, rather than an empirical force field. We apply a semi-empirical quantum-mechanical energy function, PM6-DH+, coupled with the COSMO solvation model, to 29 host-guest systems with a wide range of measured binding affinities. After correction for a systematic error, which appears to derive from the treatment of polar solvation, the computed absolute binding affinities agree well with experimental measurements, with a mean error 1.6 kcal/mol and a correlation coefficient of 0.91. These calculations also delineate the contributions of various energy components, including solute energy, configurational entropy, and solvation free energy, to the binding free energies of these host-guest complexes. Comparison with our previous calculations, which used empirical force fields, point to significant differences in both the energetic and entropic components of the binding free energy. The present study demonstrates successful combination of a quantum mechanical Hamiltonian with the M2 affinity method.

Relationship between helix stability and binding affinities: molecular dynamics simulations of Bfl-1/A1-binding pro-apoptotic BH3 peptide helices in explicit solvent.

PubMed

Modi, Vivek; Lama, Dilraj; Sankararamakrishnan, Ramasubbu

2013-01-01

The anti-apoptotic protein Bfl-1, also known as A1, belongs to the Bcl-2 family of proteins and interacts with pro-apoptotic Bcl-2 counterparts to regulate programmed cell death. As demonstrated for other anti-apoptotic Bcl-2 proteins, Bfl-1/A1 has also been shown to be overexpressed in various human cancers and hence they are attractive targets for anticancer drugs. Peptides derived from the BH3 region of pro-apoptotic Bcl-2 proteins have been shown to elicit similar biological response as that of parent proteins. BH3 peptides from different pro-apoptotic proteins have wide range of affinities for Bfl-1/A1. Experimentally determined complex structures show that the hydrophobic side of amphipathic BH3 peptides binds to the hydrophobic groove formed by the α-helical bundle of Bfl-1/A1 protein. Apart from the length and amino acid composition, a BH3 peptide's ability to form a stable helical structure has been suggested to be important for its high binding affinity. Molecular dynamics simulations of three BH3 peptides derived from the pro-apoptotic proteins Bak, Bid, and Bmf were carried out each for a period of at least 100 ns after 2 ns equilibration run. The length of simulated BH3 peptides varied from 22 to 24 residues and their binding affinities for Bfl-1/A1 varied from 1 to 180 nM. Our results show that the hydrophobic residues from the hydrophobic face of BH3 peptides tend to cluster together quickly to avoid being exposed to the solvent. This resulted in either reduction of helix length or complete loss of helical character. Bak and Bid BH3 peptides with high affinities for Bf1-1/A1 have stable helical segments in the N-terminal region. The highly conserved Leu residue lies just outside the helical region at the C-terminal end. Capping interactions arising out of N-cap residues seem to be extremely important to maintain the helical stability. Favorable hydrophilic interactions between residues also give further stability to the helix fragment and at least one of the interacting residues resides within the helical region. Bmf BH3 peptide with a weaker binding affinity for Bmf-1/A1 completely lost its helical character at the end of 100 ns production run and a further 50 ns simulation showed that the Bmf peptide continues to remain in random conformation. The present study clearly establishes a link between a BH3 peptide's ability to form a stable helical segment and its high binding affinity for an anti-apoptotic protein. To further test this hypothesis, we simulated a mutant Bmf peptide for 100 ns in which two residues R129 and H146 were substituted by Asn in silico in the wild-type peptide. Introduction of N-terminal Asn clearly enabled the formation of capping interactions at the N-terminus and resulted in a stable N-terminal helical segment. This demonstrates that the knowledge of interactions that help to maintain stable helical segments in a high-affinity BH3 peptide will help in designing highly specific peptide-based drugs/inhibitors. Such molecules will have the ability to bind a particular anti-apoptotic protein with high affinity.

A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

PubMed Central

Rao, Marie Luise; Rao, Govind S.

1982-01-01

1. Binding of l-tri-[125I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25°C. 2. The l-tri-[125I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100°C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at −38°C for several months. 3. It displayed saturability, high affinity (apparent Kd 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3′-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[125I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304±0.11μg/mg of cytosol protein (mean±s.d., n=4) was obtained; the mean concentration in plasma was 0.309±0.07μg/mg of plasma protein (mean±s.d., n=3). 7. The Ka value of 6.3×108m−1 of l-tri-[125I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[125I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin. PMID:6289813

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

PubMed Central

Gauer, Jacob W.; Knutson, Kristofer J.; Jaworski, Samantha R.; Rice, Anne M.; Rannikko, Anika M.; Lentz, Barry R.; Hinderliter, Anne

2013-01-01

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response. PMID:23746516

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

PubMed Central

Hay, Iain D.; Du, Jinping; Burr, Natalie

2014-01-01

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

Binding free energy prediction in strongly hydrophobic biomolecular systems.

PubMed

Charlier, Landry; Nespoulous, Claude; Fiorucci, Sébastien; Antonczak, Serge; Golebiowski, Jérome

2007-11-21

We present a comparison of various computational approaches aiming at predicting the binding free energy in ligand-protein systems where the ligand is located within a highly hydrophobic cavity. The relative binding free energy between similar ligands is obtained by means of the thermodynamic integration (TI) method and compared to experimental data obtained through isothermal titration calorimetry measurements. The absolute free energy of binding prediction was obtained on a similar system (a pyrazine derivative bound to a lipocalin) by TI, potential of mean force (PMF) and also by means of the MMPBSA protocols. Although the TI protocol performs poorly either with an explicit or an implicit solvation scheme, the PMF calculation using an implicit solvation scheme leads to encouraging results, with a prediction of the binding affinity being 2 kcal mol(-1) lower than the experimental value. The use of an implicit solvation scheme appears to be well suited for the study of such hydrophobic systems, due to the lack of water molecules within the binding site.

Degenerate Pax2 and Senseless binding motifs improve detection of low-affinity sites required for enhancer specificity

PubMed Central

Zandvakili, Arya; Campbell, Ian; Weirauch, Matthew T.

2018-01-01

Cells use thousands of regulatory sequences to recruit transcription factors (TFs) and produce specific transcriptional outcomes. Since TFs bind degenerate DNA sequences, discriminating functional TF binding sites (TFBSs) from background sequences represents a significant challenge. Here, we show that a Drosophila regulatory element that activates Epidermal Growth Factor signaling requires overlapping, low-affinity TFBSs for competing TFs (Pax2 and Senseless) to ensure cell- and segment-specific activity. Testing available TF binding models for Pax2 and Senseless, however, revealed variable accuracy in predicting such low-affinity TFBSs. To better define parameters that increase accuracy, we developed a method that systematically selects subsets of TFBSs based on predicted affinity to generate hundreds of position-weight matrices (PWMs). Counterintuitively, we found that degenerate PWMs produced from datasets depleted of high-affinity sequences were more accurate in identifying both low- and high-affinity TFBSs for the Pax2 and Senseless TFs. Taken together, these findings reveal how TFBS arrangement can be constrained by competition rather than cooperativity and that degenerate models of TF binding preferences can improve identification of biologically relevant low affinity TFBSs. PMID:29617378

LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

PubMed

Clayton, R N; Shakespear, R A; Marshall, J C

1978-06-01

Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

PubMed

Kaplan, Jenifer B; Reinke, Aaron W; Keating, Amy E

2014-07-01

The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides. © 2014 The Protein Society.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Predicting MHC-II binding affinity using multiple instance regression

PubMed Central

EL-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

2011-01-01

Reliably predicting the ability of antigen peptides to bind to major histocompatibility complex class II (MHC-II) molecules is an essential step in developing new vaccines. Uncovering the amino acid sequence correlates of the binding affinity of MHC-II binding peptides is important for understanding pathogenesis and immune response. The task of predicting MHC-II binding peptides is complicated by the significant variability in their length. Most existing computational methods for predicting MHC-II binding peptides focus on identifying a nine amino acids core region in each binding peptide. We formulate the problems of qualitatively and quantitatively predicting flexible length MHC-II peptides as multiple instance learning and multiple instance regression problems, respectively. Based on this formulation, we introduce MHCMIR, a novel method for predicting MHC-II binding affinity using multiple instance regression. We present results of experiments using several benchmark datasets that show that MHCMIR is competitive with the state-of-the-art methods for predicting MHC-II binding peptides. An online web server that implements the MHCMIR method for MHC-II binding affinity prediction is freely accessible at http://ailab.cs.iastate.edu/mhcmir. PMID:20855923

Accurate and sensitive quantification of protein-DNA binding affinity.

PubMed

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Accurate and sensitive quantification of protein-DNA binding affinity

PubMed Central

Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

2018-01-01

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

Identification of a receptor for ADP on blood platelets by photoaffinity labelling.

PubMed Central

Cristalli, G; Mills, D C

1993-01-01

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase. Images Figure 5 PMID:8387782

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

Differences in serotonin transporter binding affinity in patients with major depressive disorder and night eating syndrome.

PubMed

Lundgren, J D; Amsterdam, J; Newberg, A; Allison, K C; Wintering, N; Stunkard, A J

2009-03-01

We examined serotonin transporter (SERT) binding affinity using single photon emission computed tomography (SPECT) in patients with major depressive disorder (MDD) and night eating syndrome (NES). There are similarities between MDD and NES in affective symptoms, appetite disturbance, nighttime awakenings, and, particularly, response to selective serotonin reuptake inhibitors (SSRIs). Six non-depressed patients with NES and seven patients with MDD underwent SPECT brain imaging with 123I-ADAM, a radiopharmaceutical agent selective for SERT sites. Uptake ratios of 123I-ADAM SERT binding were obtained for the midbrain, basal ganglia, and temporal lobe regions compared to the cerebellum reference region. Patients with NES had significantly greater SERT uptake ratios (effect size range 0.64-0.84) in the midbrain, right temporal lobe, and left temporal lobe regions than those with MDD whom we had previously studied. Pathophysiological differences in SERT uptake between patients with NES and MDD suggest these are distinct clinical syndromes.

Warfarin resistance associated with genetic polymorphism of VKORC1: linking clinical response to molecular mechanism using computational modeling.

PubMed

Lewis, Benjamin C; Nair, Pramod C; Heran, Subash S; Somogyi, Andrew A; Bowden, Jeffrey J; Doogue, Matthew P; Miners, John O

2016-01-01

The variable response to warfarin treatment often has a genetic basis. A protein homology model of human vitamin K epoxide reductase, subunit 1 (VKORC1), was generated to elucidate the mechanism of warfarin resistance observed in a patient with the Val66Met mutation. The VKORC1 homology model comprises four transmembrane (TM) helical domains and a half helical lid domain. Cys132 and Cys135, located in the N-terminal end of TM-4, are linked through a disulfide bond. Two distinct binding sites for warfarin were identified. Site-1, which binds vitamin K epoxide (KO) in a catalytically favorable orientation, shows higher affinity for S-warfarin compared with R-warfarin. Site-2, positioned in the domain occupied by the hydrophobic tail of KO, binds both warfarin enantiomers with similar affinity. Displacement of Arg37 occurs in the Val66Met mutant, blocking access of warfarin (but not KO) to Site-1, consistent with clinical observation of warfarin resistance.

Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

PubMed

Hedlund, P B; Carson, M J; Sutcliffe, J G; Thomas, E A

1999-12-01

Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

Binding affinity and decontamination of dermal decontamination gel to model chemical warfare agent simulants.

PubMed

Cao, Yachao; Elmahdy, Akram; Zhu, Hanjiang; Hui, Xiaoying; Maibach, Howard

2018-05-01

Six chemical warfare agent simulants (trimethyl phosphate, dimethyl adipate, 2-chloroethyl methyl sulfide, diethyl adipate, chloroethyl phenyl sulfide and diethyl sebacate) were studied in in vitro human skin to explore relationship between dermal penetration/absorption and the mechanisms of simulant partitioning between stratum corneum (SC) and water as well as between dermal decontamination gel (DDGel) and water. Both binding affinity to and decontamination of simulants using DDGel were studied. Partition coefficients of six simulants between SC and water (Log P SC/w ) and between DDGel and water (Log P DDGel/w ) were determined. Results showed that DDGel has a similar or higher binding affinity to each simulant compared to SC. The relationship between Log P octanol/water and Log P SC/w as well as between Log P octanol/water and Log P DDGel/w demonstrated that partition coefficient of simulants correlated to their lipophilicity or hydrophilicity. Decontamination efficiency results with DDGel for these simulants were consistent with binding affinity results. Amounts of percentage dose of chemicals in DDGel of trimethyl phosphate, dimethyl adipate, 2-chloroethyl methyl sulfide, diethyl adipate, chloroethyl phenyl sulfide and diethyl sebacate were determined to be 61.15, 85.67, 75.91, 53.53, 89.89 and 76.58, with corresponding amounts absorbed in skin of 0.96, 0.65, 1.68, 0.72, 0.57 and 1.38, respectively. In vitro skin decontamination experiments coupled with a dermal absorption study demonstrated that DDGel can efficiently remove chemicals from skin surface, back-extract from the SC, and significantly reduced chemical penetration into skin or systemic absorption for all six simulants tested. Therefore, DDGel offers a great potential as a NextGen skin Decon platform technology for both military and civilian use. Copyright © 2018 John Wiley & Sons, Ltd.

Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

PubMed Central

Turke, Miah; Subhramanyam, Udaya K. Tiruttani; Churchill, Beth; Labahn, Joerg

2018-01-01

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists. PMID:29342106

The crystal structure of ribonuclease A in complex with thymidine-3'-monophosphate provides further insight into ligand binding.

PubMed

Doucet, Nicolas; Jayasundera, Thusitha B; Simonović, Miljan; Loria, J Patrick

2010-08-15

Thymidine-3'-monophosphate (3'-TMP) is a competitive inhibitor analogue of the 3'-CMP and 3'-UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3'-TMP binds the enzyme with a dissociation constant (K(d)) of 15 microM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3'-TMP at 1.55 A resolution and we have performed NMR binding experiments with 3'-CMP and 3'-TMP. Our results show that binding of 3'-TMP is very similar to other natural and non-natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2'-hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme. 2010 Wiley-Liss, Inc.

Let's get specific: the relationship between specificity and affinity.

PubMed

Eaton, B E; Gold, L; Zichi, D A

1995-10-01

The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.

Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity: in silico bioinformatic step-by-step guide using quantitative structure-activity relationships.

PubMed

Hattotuwagama, Channa K; Doytchinova, Irini A; Flower, Darren R

2007-01-01

Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made are freely available online at the URL http://www.jenner.ac.uk/MHCPred.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.

The transformed glucocorticoid receptor has a lower steroid-binding affinity than the nontransformed receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Takayuki; Ohara-Nemoto, Yuko; Denis, M.

1990-02-20

High-salt treatment of cytosolic glucocorticoid receptor (GR) preparations reduces the steroid-binding ability of the receptor and induces the conversion of the receptor from a nontransformed (non-DNA-binding) 9S form to a transformed (DNA-binding) 4S entity. Therefore, the authors decided to investigate the possible relationship between these two phenomena. The binding of ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) to the 9S form was almost saturated at a concentration of 20 nM, whereas ({sup 3}H)TA was hardly bound to the 4S form at this concentration. The 4S form was efficiently labeled at 200 nM. Scatchard analysis of the GR showed the presence of twomore » types of binding sites. In the absence of molybdate, the ratio of the lower affinity site was increased, but the total number of binding sites was not modified. The GR with the low ({sup 3}H)TA-binding affinity bound to DNA-cellulose even in its unliganded state, whereas the form with the high affinity did not. These results indicate that the transformed GR has a reduced ({sup 3}H)TA-binding affinity as compared to the nontransformed GR. The steroid-binding domain (amino acids 477-777) and the DNA- and steroid-binding domains (amino acids 415-777) of the human GR were expressed in Escherichia coli as protein A fused proteins. Taken together, these results suggest that the component(s) associating with the nontransformed GR, possibly the heat shock protein hsp 90, play(s) an important role in stabilizing the GR in a high-affinity state for steroids.« less

Determining ERβ Binding Affinity to Singly Mutant ERE Using Dual Polarization Interferometry

NASA Astrophysics Data System (ADS)

Song, Hong Yan; Su, Xiaodi

In a classic mode of estrogen action, estrogen receptors (ERs) bind to estrogen responsive element (ERE) to activate gene transcription. A perfect ERE contains a 13-base pair sequence of a palindromic repeat separated by a three-base spacer, 5‧-GGTCAnnnTGACC-3‧. In addition to the consensus or wild-type ERE (wtERE), naturally occurring EREs often have one or two base pairs’ alternation. Based on the newly constructed Thermodynamic Modeling of ChIP-seq (TherMos) model, binding energy between ERβ and a series of 34-bp mutant EREs (mutERE) was simulated to predict the binding affinity between ERs and EREs with single base pair deviation at different sites of the 13-bp inverted sequence. Experimentally, dual polarization interferometry (DPI) method was developed to measure ERβ-mutEREs binding affinity. On a biotin-NeutrAvidin (NA)-biotin treated DPI chip, wtERE is immobilized. In a direct binding assay, ERβ-wtERE binding affinity is determined. In a competition assay, ERβ was preincubated with mutant EREs before being added for competitive binding to the immobilized wtERE. This competition strategy provided a successful platform to evaluate the binding affinity variation among large number of ERE with different base mutations. The experimental result correlates well with the mathematically predicted binding energy with a Spearman correlation coefficient of 0.97.

Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahy, N.; Woolkalis, M.; Thermos, K.

1988-08-01

The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog (125I)CGP 23996. (125I)CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was tomore » a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity (125I)CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of (125I)CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect (125I)CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with (125I) CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to (125I)CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors.« less

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

2016-10-01

The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. PMID:28451157

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

PubMed

Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

2013-10-29

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Spatial location of neutralizing and non-neutralizing B cell epitopes on domain 1 of ricin toxin's binding subunit.

PubMed

Rong, Yinghui; Van Slyke, Greta; Vance, David J; Westfall, Jennifer; Ehrbar, Dylan; Mantis, Nicholas J

2017-01-01

Ricin toxin's binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB's high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB's high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α.

The SAMPL4 host-guest blind prediction challenge: an overview.

PubMed

Muddana, Hari S; Fenley, Andrew T; Mobley, David L; Gilson, Michael K

2014-04-01

Prospective validation of methods for computing binding affinities can help assess their predictive power and thus set reasonable expectations for their performance in drug design applications. Supramolecular host-guest systems are excellent model systems for testing such affinity prediction methods, because their small size and limited conformational flexibility, relative to proteins, allows higher throughput and better numerical convergence. The SAMPL4 prediction challenge therefore included a series of host-guest systems, based on two hosts, cucurbit[7]uril and octa-acid. Binding affinities in aqueous solution were measured experimentally for a total of 23 guest molecules. Participants submitted 35 sets of computational predictions for these host-guest systems, based on methods ranging from simple docking, to extensive free energy simulations, to quantum mechanical calculations. Over half of the predictions provided better correlations with experiment than two simple null models, but most methods underperformed the null models in terms of root mean squared error and linear regression slope. Interestingly, the overall performance across all SAMPL4 submissions was similar to that for the prior SAMPL3 host-guest challenge, although the experimentalists took steps to simplify the current challenge. While some methods performed fairly consistently across both hosts, no single approach emerged as consistent top performer, and the nonsystematic nature of the various submissions made it impossible to draw definitive conclusions regarding the best choices of energy models or sampling algorithms. Salt effects emerged as an issue in the calculation of absolute binding affinities of cucurbit[7]uril-guest systems, but were not expected to affect the relative affinities significantly. Useful directions for future rounds of the challenge might involve encouraging participants to carry out some calculations that replicate each others' studies, and to systematically explore parameter options.

T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

PubMed Central

Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

2017-01-01

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

Target-molecule-triggered rupture of aptamer-encapsulated polyelectrolyte microcapsules.

PubMed

Zhang, Xueru; Chabot, Denise; Sultan, Yasir; Monreal, Carlos; DeRosa, Maria C

2013-06-26

Polyelectrolyte microcapsules have great potential for serving as carriers for the delivery of their contents when triggered by an external stimulus. Aptamers are synthetic ssDNA or RNA that can bind to specific targets with high affinity and selectivity. Aptamers may retain these superior molecular recognition properties after encapsulation within polymer microcapsules. In this work, stable polyelectrolyte microcapsules with encapsulated aptamers were obtained by the layer-by-layer (LbL) method. Polyelectrolyte films were deposited onto a CaCO3 template that had been predoped with polystyrene sulfonate (PSS) and aptamer sequences (SA) that have an affinity for the dye sulforhodamine B (SRB). The PSS and aptamers are thought to serve as an internal scaffold supporting the microcapsule walls. These microcapsules would present target-molecule-triggered rupture properties. Microcapsule collapse was triggered by the binding of SRB to the encapsulated aptamer. The specificity of microcapsule collapse was investigated using a similar dye, tetramethylrosamine (TMR), which does not have affinity for SA. A high concentration of TMR did not lead to the collapse of the microcapsules. The effect of target binding on the microcapsules was confirmed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). These microcapsules may have potential applications in targeted delivery systems for the controlled release of drugs, pesticides, or other payloads.

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Gibberellin Receptor GID1: Gibberellin Recognition and Molecular Evolution

NASA Astrophysics Data System (ADS)

Kato, Hiroaki; Sato, Tomomi; Ueguchi-Tanaka, Miyako

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. We analyzed the crystal structure of a nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF 1 (GID1) from Oryza sativa. As it was proposed from the sequence similarity, the overall structure of GID1 shows an α/β-hydrolase fold similar to that of the hormone-sensitive lipases (HSLs) except for an amino-terminal lid. The GA-binding site corresponds to the substrate-binding site of HSLs. Almost residues assigned for GA binding showed very little or no activity when they were replaced with Ala. The substitution of the residues corresponding to those of the lycophyte GID1s caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These findings indicate that GID1 originated from HSL and was tinkered to have the specificity for bioactive GAs in the course of plant evolution.

Expanding RNA binding specificity and affinity of engineered PUF domains.

PubMed

Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-05-18

Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way.

Expanding RNA binding specificity and affinity of engineered PUF domains

PubMed Central

Zhao, Yang-Yang; Zhang, Wen-Jing; Wang, Jue; Li, Hai-Tao; Yang, Yi; Wang, Zefeng; Wu, Jia-Wei

2018-01-01

Abstract Specific manipulation of RNA is necessary for the research in biotechnology and medicine. The RNA-binding domains of Pumilio/fem-3 mRNA binding factors (PUF domains) are programmable RNA binding scaffolds used to engineer artificial proteins that specifically modulate RNAs. However, the native PUF domains generally recognize 8-nt RNAs, limiting their applications. Here, we modify the PUF domain of human Pumilio1 to engineer PUFs that recognize RNA targets of different length. The engineered PUFs bind to their RNA targets specifically and PUFs with more repeats have higher binding affinity than the canonical eight-repeat domains; however, the binding affinity reaches the peak at those with 9 and 10 repeats. Structural analysis on PUF with nine repeats reveals a higher degree of curvature, and the RNA binding unexpectedly and dramatically opens the curved structure. Investigation of the residues positioned in between two RNA bases demonstrates that tyrosine and arginine have favored stacking interactions. Further tests on the availability of the engineered PUFs in vitro and in splicing function assays indicate that our engineered PUFs bind RNA targets with high affinity in a programmable way. PMID:29490074

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C

PubMed Central

Brandoli, Giulia; Lempinen, Antti; Artes, Sanna; Turku, Ainoleena; Jäntti, Maria Helena; Talman, Virpi; Yli-Kauhaluoma, Jari; Tuominen, Raimo K.; Boije af Gennäs, Gustav

2018-01-01

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain–targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC. PMID:29641588

Scaffold hopping from (5-hydroxymethyl) isophthalates to multisubstituted pyrimidines diminishes binding affinity to the C1 domain of protein kinase C.

PubMed

Provenzani, Riccardo; Tarvainen, Ilari; Brandoli, Giulia; Lempinen, Antti; Artes, Sanna; Turku, Ainoleena; Jäntti, Maria Helena; Talman, Virpi; Yli-Kauhaluoma, Jari; Tuominen, Raimo K; Boije Af Gennäs, Gustav

2018-01-01

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain-targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, Nai-Wei; Gao, Yong; Schill, Megan S.

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirusencoded protein vCCI, a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes, and may represent a potent method to stop inflammation. Previously, our structure of the vCCI:MIP-1β complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1βN-terminus, 20’s region and 40’s loop. However, the interactionsmore » between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), another CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI:MIP-1βcomplex, and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin. Compared to wild-type eotaxin, single, double, or triple mutations at these critical charged residues weaken the binding. One exception is the K47A mutation that exhibits increased affinity for vCCI, which can be explained structurally. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1, MIP-1β and RANTES, were determined as 1.09 nM, 1.16 nM, and 0.22 nM, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and different CC chemokines.« less

Structural Analysis of Charge Discrimination in the Binding of Inhibitors to Human Carbonic Anhydrases I and II

PubMed Central

Srivastava, D. K.; Jude, Kevin M.; Banerjee, Abir L.; Haldar, Manas; Manokaran, Sumathra; Kooren, Joel; Mallik, Sanku; Christianson, David W.

2008-01-01

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively-charged, negatively-charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the 15 Å deep active site cleft, where interactions with enzyme residues and solvent molecules are possible. We find that a para-substituted positively-charged amino group is more poorly tolerated in the active site of CA I compared with CA II. In contrast, a para-substituted negatively-charged carboxylate substituent is tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail group in “two-prong” CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site desolvation and the conformational entropy of inhibitor binding to each isozyme active site. PMID:17407288

Affinities of penicillins and cephalosporins for the penicillin-binding proteins of Escherichia coli K-12 and their antibacterial activity.

PubMed Central

Curtis, N A; Orr, D; Ross, G W; Boulton, M G

1979-01-01

The affinities of a range of penicillins and cephalosporins for ther penicillin-binding proteins of Escherichia coli K-12 have been studied, and the results were compared with the antibacterial activity of the compounds against E. coli K-12 and an isogenic permeability mutant. Different penicillins and cephalosporins exhibited different affinities for the "essential" penicillin-binding proteins of E. coli K-12, in a manner which directly correlated with their observed effects upon bacterial morphology. Furthermore, the affinities of the compounds for their "primary" lethal penicillin-binding protein targets showed close agreement with their antibacterial activities against the permeability mutant. Images PMID:393164

Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

NASA Astrophysics Data System (ADS)

Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

2008-05-01

Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

Isolation and characterization of fatty acid binding protein in the liver of the nurse shark, Ginglymostoma cirratum.

PubMed

Bass, N M; Manning, J A; Luer, C A

1991-01-01

1. A 14.5 kDa fatty acid binding protein was isolated from the liver of the nurse shark, Ginglymostoma cirratum. 2. Purified shark liver FABP (pI = 5.4) bound oleic acid at a single site with an affinity similar to that of mammalian FABP. 3. The apparent size, pI and amino acid composition of shark liver FABP indicate a close structural relationship between this protein and mammalian heart FABP.

A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide.

PubMed

Wessels, M R; Muñoz, A; Kasper, D L

1987-12-01

We recently reported that the single repeating-unit pentasaccharide of type III group B Streptococcus (GBS) capsular polysaccharide is only weakly reactive with type III GBS antiserum. To further elucidate the relationship between antigen-chain length and antigenicity, tritiated oligosaccharides derived from type III capsular polysaccharide were used to generate detailed saturation binding curves with a fixed concentration of rabbit antiserum in a radioactive antigen-binding assay. A graded increase in affinity of antigen-antibody binding was seen as oligosaccharide size increased from 2.6 repeating units to 92 repeating units. These differences in affinity of antibody binding to oligosaccharides of different molecular size were confirmed by immunoprecipitation and competitive ELISA, two independent assays of antigen-antibody binding. Analysis of the saturation binding experiment indicated a difference of 300-fold in antibody-binding affinity for the largest versus the smallest tested oligosaccharides. Unexpectedly, the saturation binding values approached by the individual curves were inversely related to oligosaccharide chain length on a molar basis but equivalent on a weight basis. This observation is compatible with a model in which binding of an immunoglobulin molecule to an antigenic site on the polysaccharide facilitates subsequent binding of antibody to that antigen.

Increased thermolability of benzodiazepine receptors in cerebral cortex of a baboon with spontaneous seizures: a case report.

PubMed

Squires, R; Naquet, R; Riche, D; Braestrup, C

1979-06-01

The benzodiazepine receptor in the cortex of 1 spontaneously epileptic baboon exhibited an increased rate of thermal inactivation at 65 degrees C when compared with those from 3 other baboons. In other respects (receptor concentration, affinities for flunitrazepam and diazepam, and response to changing pH), the benzodiazepine receptor from this animal was very similar to the receptors in the cortex of 3 other baboons. The 3H-QNB (muscarinic) and 3H-naloxone (opiate) binding sites in the brain of all 4 baboons appeared very similar with respect to all parameters studied (thermal stability, concentration, regional distribution, and affinities for respective ligands). An endogenous factor stabilizing the benzodiazepine receptor could be lacking in the spontaneously epileptic baboon.

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

PubMed

Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

2015-01-01

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities

PubMed Central

Petrič, Andrej; Johnson, Scott A.; Pham, Hung V.; Li, Ying; Čeh, Simon; Golobič, Amalija; Agdeppa, Eric D.; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N.; Barrio, Jorge R.

2012-01-01

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. PMID:23012452

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

PubMed

Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

2012-10-12

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module*

PubMed Central

Taylor, Courtney B.; Talib, M. Faiz; McCabe, Clare; Bu, Lintao; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3–6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function. PMID:22147693

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

Biochemical study of prolactin binding sites in Xenopus laevis brain and choroid plexus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muccioli, G.; Guardabassi, A.; Pattono, P.

1990-03-01

The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrencemore » of prolactin specific binding sites in Xenopus laevis choroid plexus cells.« less

Evolution of a Histone H4-K16 Acetyl-Specific DNA Aptamer

PubMed Central

Williams, Berea A. R.; Lin, Liyun; Lindsay, Stuart M.; Chaput, John C.

2009-01-01

We report the in vitro selection of DNA aptamers that bind to histone H4 proteins acetylated at lysine 16. The best aptamer identified in this selection binds to the target protein with a Kd of 21 nM, and discriminates against both the non-acetylated protein and histone H4 proteins acetylated at lysine 8. Comparative binding assays performed with a chip-quality antibody reveal that this aptamer binds to the acetylated histone target with similar affinity to a commercial antibody, but shows significantly greater specificity (15-fold versus 2,400-fold) for the target molecule. This result demonstrates that aptamers that are both modification and location specific can be generated to bind specific protein post-translational modifications. PMID:19385619

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

PubMed Central

Bai, Erdeni; Rosell, Federico I.; Lige, Bao; Mauk, Marcia R.; Lelj-Garolla, Barbara; Moore, Geoffrey R.; Mauk, A. Grant

2006-01-01

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents. PMID:16928194

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

PubMed

Berry, L M; Adams, R; Airey, M; Bracher, M G; Bourne, T; Carrington, B; Cross, A S; Davies, G C G; Finney, H M; Foulkes, R; Gozzard, N; Griffin, R A; Hailu, H; Lamour, S D; Lawson, A D; Lightwood, D J; McKnight, A J; O'Dowd, V L; Oxbrow, A K F; Popplewell, A G; Shaw, S; Stephens, P E; Sweeney, B; Tomlinson, K L; Uhe, C; Palframan, R T

2009-02-01

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74.

PubMed

Miller, Philip S; Barwell, James; Poyner, David R; Wigglesworth, Mark J; Garland, Stephen L; Donnelly, Dan

2010-01-01

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1. Copyright 2009 Elsevier Inc. All rights reserved.

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

PubMed Central

Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

2011-01-01

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1.

PubMed

Real Hernandez, Luis M; Gonzalez de Mejia, Elvira

2017-04-01

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.

Botulinum neurotoxin G binds synaptotagmin-II in a mode similar to that of serotype B: tyrosine 1186 and lysine 1191 cause its lower affinity.

PubMed

Willjes, Gesche; Mahrhold, Stefan; Strotmeier, Jasmin; Eichner, Timo; Rummel, Andreas; Binz, Thomas

2013-06-04

Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoforms I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for mosaic-type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B-synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information about BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased the level of binding of BoNT/G approximately to the level of BoNT/B affinity, suggesting a similar synaptotagmin-bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. On the basis of these data, molecular modeling experiments were employed to reveal an atomistic model of BoNT/G-synaptotagmin recognition. These data suggest a reduced length and/or a bend in the C-terminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses.

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

PubMed Central

Zhao, Wenjing; McCallum, Scott A.; Xiao, Zhongping; Zhang, Fuming; Linhardt, Robert J.

2011-01-01

Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs. PMID:21658003

Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

PubMed Central

1984-01-01

Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites. PMID:6088662

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

PubMed

Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

2014-11-18

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

PubMed Central

Wijeyesakere, Sanjeeva Joseph; Gagnon, Jessica K.; Arora, Karunesh; Brooks, Charles L.; Raghavan, Malini

2015-01-01

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics. PMID:26420867

Determinants of the Differential Antizyme-Binding Affinity of Ornithine Decarboxylase

PubMed Central

Liu, Yen-Chin; Hsu, Den-Hua; Huang, Chi-Liang; Liu, Yi-Liang; Liu, Guang-Yaw; Hung, Hui-Chih

2011-01-01

Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K d value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K d value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K d was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K d, which suggests that residues 119 and 137 play a role in AZ binding. PMID:22073206

Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

PubMed

Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

1989-07-01

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.

Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites.

PubMed Central

Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G

1989-01-01

Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin. PMID:2544892

Structure-activity relationships studies on weakly basic N-arylsulfonylindoles with an antagonistic profile in the 5-HT6 receptor

NASA Astrophysics Data System (ADS)

Mella, Jaime; Villegas, Francisco; Morales-Verdejo, César; Lagos, Carlos F.; Recabarren-Gajardo, Gonzalo

2017-07-01

We recently reported a series of 39 weakly basic N-arylsulfonylindoles as novel 5-HT6 antagonists. Eight of the compounds exhibited moderate to high binding affinities, with 2-(4-(2-Methoxyphenyl)piperazin-1-yl)-1-(1-tosyl-1H-indol-3-yl)ethanol 16 showing the highest binding affinity (pKi = 7.87). Given these encouraging results and as a continuation of our research, we performed an extensive step-by-step search for the best 3D-QSAR model that allows us to rationally propose novel molecules with improved 5-HT6 affinity based on our previously reported series. A comparative molecular similarity indices analysis (CoMSIA) model built on a docking-based alignment was developed, wherein steric, electrostatic, hydrophobic and hydrogen bond properties are correlated with biological activity. The model was validated internally and externally (q2 = 0.721; r2pred = 0.938), and identified the sulfonyl and hydroxyl groups and the piperazine ring among the main regions of the molecules that can be modified to create new 5-HT6 antagonists.

Measurement of radon and xenon binding to a cryptophane molecular host

PubMed Central

Jacobson, David R.; Khan, Najat S.; Collé, Ronald; Fitzgerald, Ryan; Laureano-Pérez, Lizbeth; Bai, Yubin; Dmochowski, Ivan J.

2011-01-01

Xenon and radon have many similar properties, a difference being that all 35 isotopes of radon (195Rn–229Rn) are radioactive. Radon is a pervasive indoor air pollutant believed to cause significant incidence of lung cancer in many geographic regions, yet radon affinity for a discrete molecular species has never been determined. By comparison, the chemistry of xenon has been widely studied and applied in science and technology. Here, both noble gases were found to bind with exceptional affinity to tris-(triazole ethylamine) cryptophane, a previously unsynthesized water-soluble organic host molecule. The cryptophane–xenon association constant, Ka = 42,000 ± 2,000 M-1 at 293 K, was determined by isothermal titration calorimetry. This value represents the highest measured xenon affinity for a host molecule. The partitioning of radon between air and aqueous cryptophane solutions of varying concentration was determined radiometrically to give the cryptophane–radon association constant Ka = 49,000 ± 12,000 M-1 at 293 K. PMID:21690357

SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G

PubMed Central

Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P.; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

2017-01-01

The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis. PMID:28401063

THE EFFECTS OF TYPE II BINDING ON METABOLIC STABILITY AND BINDING AFFINITY IN CYTOCHROME P450 CYP3A4

PubMed Central

Peng, Chi-Chi; Pearson, Josh T.; Rock, Dan A.; Joswig-Jones, Carolyn A.; Jones, Jeffrey P.

2010-01-01

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound’s metabolic stability can be increased by incorporation of a sp2 nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1,200 fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding. PMID:20346909

Pharmacological characterization of the bradykinin B2 receptor: inter-species variability and dissociation between binding and functional responses

PubMed Central

Paquet, J -L; Luccarini, J -M; Fouchet, C; Defrêne, E; Loillier, B; Robert, C; Bélichard, P; Cremers, B; Pruneau, D

1999-01-01

The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1-oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer. BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively). In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter-species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents. PMID:10204994

New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

PubMed Central

Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

2013-01-01

3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

Endothelin mechanisms in altered thyroid states in the rat.

PubMed

Rebello, S; Thompson, E B; Gulati, A

1993-06-11

Endothelin (ET) and its receptor characteristics were studied in hyper- and hypo-thyroid states in the rats. Hyperthyroidism was induced by daily administration of thyroxine (0.1 mg/kg i.p.) for 8 weeks, while hypothyrodism was induced by daily administration of methimazole (10 mg/kg i.p.) for 8 weeks. The chronic administration of thyroxine to rats decreased their rate of gain of body weight, increased serum T3 and T4 concentration, blood pressure and heart rate. The chronic administration of methimazole decreased the rate of gain of body weight, serum T3 and T4 concentration, blood pressure and heart rate as compared to vehicle-treated control. Plasma ET-1 levels were found to be similar in control and methimazole-treated rats, while the levels were found to be significantly (P < 0.002) increased in thyroxine-treated rats as compared to control rats. Binding studies showed that [125I]ET-1 bound to a single, high affinity binding site in the cerebral cortex, hypothalamus and pituitary. The density (Bmax) and the affinity (Kd) of [125I]ET-1 binding in the cerebral cortex and hypothalamus were found to be similar in control, methimazole- and thyroxine-treated rats. The pituitary of thyroxine-treated rats showed a decrease in the binding (34.3% decrease in the density) of [125I]ET-1 as compared to control rats. No difference was observed in the binding of [125I]ET-1 to pituitary membranes from control and methimazole-treated rats. Competition studies showed that the IC50 and Ki values of ET-3 for [125]ET-1 binding were about 8 to 11 times higher than ET-1 in cerebral cortex, hypothalamus and pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a Gene Family Encoding SEA (Sea-urchin Sperm Protein, Enterokinase and Agrin)-Domain Proteins with Lectin-Like and Heme-Binding Properties from Schistosoma japonicum

PubMed Central

Mbanefo, Evaristus Chibunna; Kikuchi, Mihoko; Huy, Nguyen Tien; Shuaibu, Mohammed Nasir; Cherif, Mahamoud Sama; Yu, Chuanxin; Wakao, Masahiro; Suda, Yasuo; Hirayama, Kenji

2014-01-01

Background We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. Methodology/Principal Findings Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10−6 M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. Conclusions The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted. PMID:24416467

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions

PubMed Central

Shukla, Suneet; Abel, Biebele; Chufan, Eduardo E.; Ambudkar, Suresh V.

2017-01-01

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10–40 nm range. Similarly, a 30–150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment. PMID:28283574

The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

PubMed

Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

2015-12-01

Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation. © 2015 International Society for Neurochemistry.

Relative binding affinities of monolignols to horseradish peroxidase

DOE PAGES

Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; ...

2016-07-22

Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic –OH group andmore » a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic –OH group instead interacting with Pro139. Furthermore, since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.« less

Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.

PubMed

Martí-Arbona, Ricardo; Teshima, Munehiro; Anderson, Penelope S; Nowak-Lovato, Kristy L; Hong-Geller, Elizabeth; Unkefer, Clifford J; Unkefer, Pat J

2012-01-01

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding. Copyright © 2012 S. Karger AG, Basel.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

Solubilization and purification of melatonin receptors from lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Exploring the Origin of Differential Binding Affinities of Human Tubulin Isotypes αβII, αβIII and αβIV for DAMA-Colchicine Using Homology Modelling, Molecular Docking and Molecular Dynamics Simulations

PubMed Central

Panda, Dulal; Kunwar, Ambarish

2016-01-01

Tubulin isotypes are found to play an important role in regulating microtubule dynamics. The isotype composition is also thought to contribute in the development of drug resistance as tubulin isotypes show differential binding affinities for various anti-cancer agents. Tubulin isotypes αβII, αβIII and αβIV show differential binding affinity for colchicine. However, the origin of differential binding affinity is not well understood at the molecular level. Here, we investigate the origin of differential binding affinity of a colchicine analogue N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine) for human αβII, αβIII and αβIV isotypes, employing sequence analysis, homology modeling, molecular docking, molecular dynamics simulation and MM-GBSA binding free energy calculations. The sequence analysis study shows that the residue compositions are different in the colchicine binding pocket of αβII and αβIII, whereas no such difference is present in αβIV tubulin isotypes. Further, the molecular docking and molecular dynamics simulations results show that residue differences present at the colchicine binding pocket weaken the bonding interactions and the correct binding of DAMA-colchicine at the interface of αβII and αβIII tubulin isotypes. Post molecular dynamics simulation analysis suggests that these residue variations affect the structure and dynamics of αβII and αβIII tubulin isotypes, which in turn affect the binding of DAMA-colchicine. Further, the binding free-energy calculation shows that αβIV tubulin isotype has the highest binding free-energy and αβIII has the lowest binding free-energy for DAMA-colchicine. The order of binding free-energy for DAMA-colchicine is αβIV ≃ αβII >> αβIII. Thus, our computational approaches provide an insight into the effect of residue variations on differential binding of αβII, αβIII and αβIV tubulin isotypes with DAMA-colchicine and may help to design new analogues with higher binding affinities for tubulin isotypes. PMID:27227832

Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lentz, T.L.

1991-11-12

Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similarmore » segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.« less

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2

PubMed Central

Petukh, Marharyta; Wu, Bohua; Stefl, Shannon; Smith, Nick; Hyde-Volpe, David; Wang, Li; Alexov, Emil

2014-01-01

Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade. PMID:25369028

The actions of some esters of 4-hydroxyquinuclidine on guinea-pig ileum, atria and rat fundus strip.

PubMed

Barlow, R B; Kitchen, R

1982-11-01

1 The acetyl, phenylacetyl, and diphenylacetyl esters of 4-hydroxyquinuclidine and their methiodides have been prepared.2 4-Diphenylacetoxyquinuclidine methiodide has higher affinity for muscarinic receptors than 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP methiodide) but it is less selective. At 30 degrees C its affinity for receptors in ileum is about 5 times that for receptors in atria, a difference similar to that found with diphenylacetoxytrophine methiodide. With 4-DAMP methiodide affinity for receptors in the ileum is over 10 times that for receptors in atria.3 4-Diphenylacetoxyquinuclidine methiodide has higher affinity for muscarinic receptors than 3-diphenylacetoxyquinuclidine hydrochloride or its methiodide.4 4-Acetoxyquinuclidine hydrochloride has less than one-hundredth of the activity of 3-acetoxyquinuclidine hydrochloride (acecyclidine) on guinea-pig ileum, atria, and rat fundus: however, 4-acetoxyquinuclidine methiodide is consistently more active than its 3-isomer, though it is only about 1/25 times as active as acecyclidine.5 4-Acetoxyquinuclidine hydrochloride is only a poor substrate for electric eel acetylcholinesterase: its affinity is similar to that of acecyclidine but it is greatly reduced by methylation.6 The relations between the structure and activity of the agonists are very different from the relations between the structure and affinity of the antagonists, which supports the view that agonists and antagonists bind to different conformations of the muscarinic receptor.

Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells.

PubMed

Saxenhofer, H; Fitzgibbon, W R; Paul, R V

1993-02-01

Urodilatin (URO) [ANP-(95-126)] is an analogue of atrial natriuretic peptide (alpha-ANP) [ANP-(99-126)] that was first isolated from human urine. In rat mesangial cells, URO competed with high affinity for non-guanylate cyclase-coupled ANPR-C receptors [concentration at which 50% labeled ligand is displaced (IC50) approximately 70 pM], but with lesser affinity to the guanylate cyclase-linked ANPR-A receptors (IC50 approximately 800 pM). alpha-ANP bound to both receptors with similar affinity [dissociation constant (Kd) approximately 150 pM]. In papillary collecting duct homogenates, which possess only ANPR-A receptors, the apparent Kd value averaged 229 pM for alpha-ANP and 2.7 nM for URO. Intravenous URO was at least as potent and effective as alpha-ANP in inducing diuresis and natriuresis in anesthetized rats, but URO was approximately 10-fold less potent in stimulating guanosine 3',5'-cyclic monophosphate generation in mesangial and inner medullary collecting duct cells. We conclude that URO has a lesser affinity than alpha-ANP for guanylate cyclase-coupled ANP receptors in the kidney and that the relative natriuretic potency of URO in vivo cannot be directly attributed to its binding characteristics with ANPR-A receptors.

Receptor interactrions of imidazolines. VI. Significance of carbon bridge separating phenyl and imidazoline rings of tolazoline-like alpha adrenergic imidazolines.

PubMed

Ruffolo, R R; Yaden, E L; Waddell, J E; Dillard, R D

1980-09-01

The pharmacological significance of the carbon bridge separating the imidazoline and phenyl rings of tolazoline-like alpha adrenergic imidazolines has been investigated. Extending the carbon bridge to two carbon atoms, or deleting the carbon bridge, lowers affinity of the imidazolines for the alpha receptor and markedly decreases or abolishes efficacy (i.e., agonist activity), suggesting that a single carbon atome optimallyu separates the phenyl and imidazoline rings. Although one carbon is optimal for alpha adrenergic activity, this particular atom does not appear to be essential since nitrogen may substitute for carbon with no marked or consistent changes observed in affinity or efficacy. Hydroxylation of the carbon bridge decreases affinity for the receptor approximately 10-fold but does not alter efficacy, whereas a similar substitution made in the norepinephrine-series of phenethylamines markedly increases affinity (Patil et al., 1974). With both the imidazolines and phenethylamines, this carbon atom may stereoselectively influence binding to the receptor. These results suggest that the carbon atom bridging the phenyl and imidazoline rings of tolazoline-like imidazolines serves only to provide optimal separation between these rings and does not contribute directly to the binding process. It is proposed that alpha adrenergic imidazolines interact differently with the alpha adrenergic receptor than the norepinephrine-like phenethylamines.

The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

PubMed

Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

2010-02-05

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

PubMed

Henderson, Brittney R; Saeedi, Bejan J; Campagnola, Grace; Geiss, Brian J

2011-01-01

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Tungsten Transport Protein A (WtpA) in Pyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

PubMed Central

Bevers, Loes E.; Hagedoorn, Peter-Leon; Krijger, Gerard C.; Hagen, Wilfred R.

2006-01-01

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [KD] of 17 ± 7 pM) and molybdate (KD of 11 ± 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low KD values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein. PMID:16952940

Mechanistic insights into the recognition of 5-methylcytosine oxidation derivatives by the SUVH5 SRA domain.

PubMed

Rajakumara, Eerappa; Nakarakanti, Naveen Kumar; Nivya, M Angel; Satish, Mutyala

2016-02-04

5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives.

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

PubMed

Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

2016-03-14

The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit.

PubMed Central

Ferenci, T; Lee, K S

1989-01-01

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits. PMID:2521623

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

Comparison of the Efficiency of the LIE and MM/GBSA Methods to Calculate Ligand-Binding Energies.

PubMed

Genheden, Samuel; Ryde, Ulf

2011-11-08

We have evaluated the efficiency of two popular end-point methods to calculate ligand-binding free energies, LIE (linear interaction energy) and MM/GBSA (molecular mechanics with generalized Born surface-area solvation), i.e. the computational effort needed to obtain estimates of a similar precision. As a test case, we use the binding of seven biotin analogues to avidin. The energy terms used by MM/GBSA and LIE exhibit a similar correlation time (∼5 ps), and the equilibration time seems also to be similar, although it varies much between the various ligands. The results show that the LIE method is more effective than MM/GBSA, by a factor of 2-7 for a truncated spherical system with a radius of 26 Å and by a factor of 1.0-2.4 for the full avidin tetramer (radius 47 Å). The reason for this is the cost for the MM/GBSA entropy calculations, which more than compensates for the extra simulation of the free ligand in LIE. On the other hand, LIE requires that the protein is neutralized, whereas MM/GBSA has no such requirements. Our results indicate that both the truncation and neutralization of the proteins may slow the convergence and emphasize small differences in the calculations, e.g., differences between the four subunits in avidin. Moreover, LIE cannot take advantage of the fact that avidin is a tetramer. For this test case, LIE gives poor results with the standard parametrization, but after optimizing the scaling factor of the van der Waals terms, reasonable binding affinities can be obtained, although MM/GBSA still gives a significantly better predictive index and correlation to the experimental affinities.

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

PubMed

Kane, M A; Portanova, L B; Kelley, K; Holley, M; Ross, S E; Boose, D; Escobedo-Morse, A; Alvarado, B

1994-01-01

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

PubMed

Marsh, Lorraine

2015-01-01

Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase

PubMed Central

2013-01-01

Background Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth. PMID:23621895

Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action

NASA Technical Reports Server (NTRS)

Yang, T.; Poovaiah, B. W.

2000-01-01

The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain. A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner. Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction.

Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

PubMed

Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

2016-10-01

Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. Copyright © 2016 Elsevier B.V. All rights reserved.

New opioid receptor antagonist: Naltrexone-14-O-sulfate synthesis and pharmacology.

PubMed

Zádor, Ferenc; Király, Kornél; Váradi, András; Balogh, Mihály; Fehér, Ágnes; Kocsis, Dóra; Erdei, Anna I; Lackó, Erzsébet; Zádori, Zoltán S; Hosztafi, Sándor; Noszál, Béla; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

2017-08-15

Opioid antagonists, naloxone and naltrexone have long been used in clinical practice and research. In addition to their low selectivity, they easily pass through the blood-brain barrier. Quaternization of the amine group in these molecules, (e.g. methylnaltrexone) results in negligible CNS penetration. In addition, zwitterionic compounds have been reported to have limited CNS access. The current study, for the first time gives report on the synthesis and the in vitro [competition binding, G-protein activation, isolated mouse vas deferens (MVD) and mouse colon assay] pharmacology of the zwitterionic compound, naltrexone-14-O-sulfate. Naltrexone, naloxone, and its 14-O-sulfate analogue were used as reference compounds. In competition binding assays, naltrexone-14-O-sulfate showed lower affinity for µ, δ or κ opioid receptor than the parent molecule, naltrexone. However, the μ/κ opioid receptor selectivity ratio significantly improved, indicating better selectivity. Similar tendency was observed for naloxone-14-O-sulfate when compared to naloxone. Naltrexone-14-O-sulfate failed to activate [ 35 S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile 5,6 deltorphin II and U69593), similarly to the reference compounds. Schild plot constructed in MVD revealed that naltrexone-14-O-sulfate acts as a competitive antagonist. In mouse colon, naltrexone-14-O-sulfate antagonized the inhibitory effect of morphine with lower affinity compared to naltrexone and higher affinity when compared to naloxone or naloxone-14-O-sulfate. In vivo (mouse tail-flick test), subcutaneously injected naltrexone-14-O-sulfate antagonized morphine's antinociception in a dose-dependent manner, indicating it's CNS penetration, which was unexpected from such zwitter ionic structure. Future studies are needed to evaluate it's pharmacokinetic profile. Copyright © 2017 Elsevier B.V. All rights reserved.

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

PubMed

Matulis, Daumantas; Kranz, James K; Salemme, F Raymond; Todd, Matthew J

2005-04-05

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

Selectivity of arsenite interaction with zinc finger proteins.

PubMed

Zhao, Linhong; Chen, Siming; Jia, Liangyuan; Shu, Shi; Zhu, Pingping; Liu, Yangzhong

2012-08-01

Arsenic is a carcinogenic element also used for the treatment of acute promyelocytic leukemia. The reactivity of proteins to arsenic must be associated with the various biological functions of As. Here, we investigated the selectivity of arsenite to zinc finger proteins (ZFPs) with different zinc binding motifs (C2H2, C3H, and C4). Single ZFP domain proteins were used for the direct comparison of the reactivity of different ZFPs. The binding constants and the reaction rates have been studied quantitatively. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4 > C3H ≫ C2H2. Compared with the C2H2 motif ZFPs, the binding affinities of C3H and C4 motif ZFPs are nearly two orders of magnitude higher and the reaction rates are approximately two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2 type ZFPs, but has negligible effects on C3H and C4 ZFPs. Consequently, the reactivities of the three types of multi-domain ZFPs are rather similar. The 2D NMR spectra indicate that the As(III)-bound ZFPs are also unfolded, suggesting that arsenic binding interferes with the function of ZFPs.

[Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

PubMed

Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

2012-01-01

It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

PubMed

Matsumoto, Yuki; Shindo, Yosuke; Takakusagi, Yoichi; Takakusagi, Kaori; Tsukuda, Senko; Kusayanagi, Tomoe; Sato, Hitoshi; Kawabe, Takumi; Sugawara, Fumio; Sakaguchi, Kengo

2011-12-01

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix. Copyright © 2011 Elsevier Ltd. All rights reserved.

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular and Functional Characterization of Odorant-Binding Protein Genes in an Invasive Vector Mosquito, Aedes albopictus

PubMed Central

Deng, Yuhua; Yan, Hui; Gu, Jinbao; Xu, Jiabao; Wu, Kun; Tu, Zhijian; James, Anthony A.; Chen, Xiaoguang

2013-01-01

Aedes albopictus is a major vector of dengue and Chikungunya viruses. Olfaction plays a vital role in guiding mosquito behaviors and contributes to their ability to transmit pathogens. Odorant-binding proteins (OBPs) are abundant in insect olfactory tissues and involved in the first step of odorant reception. While comprehensive descriptions are available of OBPs from Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae, only a few genes from Ae. albopictus have been reported. In this study, twenty-one putative AalbOBP genes were cloned using their homologues in Ae. aegypti to query an Ae. albopictus partial genome sequence. Two antenna-specific OBPs, AalbOBP37 and AalbOBP39, display a remarkable similarity in their overall folding and binding pockets, according to molecular modeling. Binding affinity assays indicated that AalbOBP37 and AalbOBP39 had overlapping ligand affinities and are affected in different pH condition. Electroantennagrams (EAG) and behavioral tests show that these two genes were involved in olfactory reception. An improved understanding of the Ae. albopictus OBPs is expected to contribute to the development of more efficient and environmentally-friendly mosquito control strategies. PMID:23935894

Controlled rotation of the F1-ATPase reveals differential and continuous binding changes for ATP synthesis

PubMed Central

Adachi, Kengo; Oiwa, Kazuhiro; Yoshida, Masasuke; Nishizaka, Takayuki; Kinosita, Kazuhiko

2012-01-01

F1-ATPase is an ATP-driven rotary molecular motor that synthesizes ATP when rotated in reverse. To elucidate the mechanism of ATP synthesis, we imaged binding and release of fluorescently labelled ADP and ATP while rotating the motor in either direction by magnets. Here we report the binding and release rates for each of the three catalytic sites for 360° of the rotary angle. We show that the rates do not significantly depend on the rotary direction, indicating ATP synthesis by direct reversal of the hydrolysis-driven rotation. ADP and ATP are discriminated in angle-dependent binding, but not in release. Phosphate blocks ATP binding at angles where ADP binding is essential for ATP synthesis. In synthesis rotation, the affinity for ADP increases by >104, followed by a shift to high ATP affinity, and finally the affinity for ATP decreases by >104. All these angular changes are gradual, implicating tight coupling between the rotor angle and site affinities. PMID:22929779

Water-mediated protein-fluorophore interactions modulate the affinity of an ABC-ATPase/TNP-ADP complex.

PubMed

Oswald, Christine; Jenewein, Stefan; Smits, Sander H J; Holland, I Barry; Schmitt, Lutz

2008-04-01

TNP-modified nucleotides have been used extensively to study protein-nucleotide interactions. In the case of ABC-ATPases, application of these powerful tools has been greatly restricted due to the significantly higher affinity of the TNP-nucleotide for the corresponding ABC-ATPase in comparison to the non-modified nucleotides. To understand the molecular changes occurring upon binding of the TNP-nucleotide to an ABC-ATPase, we have determined the crystal structure of the TNP-ADP/HlyB-NBD complex at 1.6A resolution. Despite the higher affinity of TNP-ADP, no direct fluorophore-protein interactions were observed. Unexpectedly, only water-mediated interactions were detected between the TNP moiety and Tyr(477), that is engaged in pi-pi stacking with the adenine ring, as well as with two serine residues (Ser(504) and Ser(509)) of the Walker A motif. Interestingly, the side chains of these two serine residues adopt novel conformations that are not observed in the corresponding ADP structure. However, in the crystal structure of the S504A mutant, which binds TNP-ADP with similar affinity to the wild type enzyme, a novel TNP-water interaction compensates for the missing serine side chain. Since this water molecule is not present in the wild type enzyme, these results suggest that only water-mediated interactions provide a structural explanation for the increased affinity of TNP-nucleotides towards ABC-ATPases. However, our results also imply that in silico approaches such as docking or modeling cannot directly be applied to generate 'affinity-adopted' ADP- or ATP-analogs for ABC-ATPases.

Sequence2Vec: a novel embedding approach for modeling transcription factor binding affinity landscape.

PubMed

Dai, Hanjun; Umarov, Ramzan; Kuwahara, Hiroyuki; Li, Yu; Song, Le; Gao, Xin

2017-11-15

An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these hidden Markov models into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA datasets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods. Our program is freely available at https://github.com/ramzan1990/sequence2vec. xin.gao@kaust.edu.sa or lsong@cc.gatech.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Concepts in receptor optimization: targeting the RGD peptide.

PubMed

Chen, Wei; Chang, Chia-en; Gilson, Michael K

2006-04-12

Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Presence of Laminin Receptors in Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Lopes, J. D.; Dos Reis, M.; Brentani, R. R.

1985-07-01

A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

Altered Enthalpy-Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase†

PubMed Central

Edwards, Achelle A.; Mason, Jennifer M.; Clinch, Keith; Tyler, Peter C.; Evans, Gary B.; Schramm, Vern L.

2009-01-01

Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetery indicate that binding of a structurally rigid first-generation Immucillin (K d = 56 pM) is driven by large negative enthalpy values (ΔH = −21.2 kcal/mol) with a substantial entropic (-TΔS) penalty. The tightest-binding inhibitors (K d = 5 to 9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP·inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5’-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the seventeen Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. By introducing flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding. PMID:19425594

Comparative theoretical study of the binding of luciferyl-adenylate and dehydroluciferyl-adenylate to firefly luciferase

NASA Astrophysics Data System (ADS)

Pinto da Silva, Luís; Vieira, João; Esteves da Silva, Joaquim C. G.

2012-08-01

This is the first report of a study employing a computational approach to study the binding of (D/L)-luciferyl-adenlyates and dehydroluciferyl-adenylate to firefly luciferase. A semi-empirical/molecular mechanics methodology was used to study the interaction between these ligands and active site molecules. All adenylates are complexed with the enzyme, mostly due to electrostatic interactions with cationic residues. Dehydroluciferyl-adenylate is expected to be a competitive inhibitor of luciferyl-adenylate, as their binding mechanism and affinity to luciferase are very similar. Both luciferyl-adenylates adopt the L-orientation in the active site of luciferase.

An additional role for the Brønsted acid-base catalysts of mandelate racemase in transition state stabilization.

PubMed

Nagar, Mitesh; Bearne, Stephen L

2015-11-10

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelate and serves as a paradigm for understanding the enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. The enzyme utilizes a two-base mechanism with Lys 166 and His 297 acting as Brønsted acid and base catalysts, respectively, in the R → S reaction direction. In the S → R reaction direction, their roles are reversed. Using isothermal titration calorimetry (ITC), MR is shown to bind the intermediate/transition state (TS) analogue inhibitor benzohydroxamate (BzH) in an entropy-driven process with a value of ΔCp equal to -358 ± 3 cal mol(-1) K(-1), consistent with an increased number of hydrophobic interactions. However, MR binds BzH with an affinity that is ∼2 orders of magnitude greater than that predicted solely on the basis of hydrophobic interactions [St. Maurice, M., and Bearne, S. L. (2004) Biochemistry 43, 2524], suggesting that additional specific interactions contribute to binding. To test the hypothesis that cation-π/NH-π interactions between the side chains of Lys 166 and His 297 and the aromatic ring and/or the hydroxamate/hydroximate moiety of BzH contribute to the binding of BzH, site-directed mutagenesis was used to generate the MR variants K166M, K166C, H297N, and K166M/H297N and their binding affinity for various ligands determined using ITC. Comparison of the binding affinities of these MR variants with the intermediate/TS analogues BzH and cyclohexanecarbohydroxamate revealed that cation-π/NH-π interactions between His 297 and the hydroxamate/hydroximate moiety and the phenyl ring of BzH contribute approximately 0.26 and 0.91 kcal/mol to binding, respectively, while interactions with Lys 166 contribute approximately 1.74 and 1.74 kcal/mol, respectively. Similarly, comparison of the binding affinities of these mutants with substrate analogues revealed that Lys 166 contributes >2.93 kcal/mol to the binding of (R)-atrolactate, and His 297 contributes 2.46 kcal/mol to the binding of (S)-atrolactate. These results are consistent with Lys 166 and His 297 playing dual roles in catalysis: they act as Brønsted acid-base catalysts, and they stabilize both the enolate moiety and phenyl ring of the altered substrate in the TS.

A Thermoacidophile-Specific Protein Family, DUF3211, Functions as a Fatty Acid Carrier with Novel Binding Mode

PubMed Central

Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi; Miyauchi, Yumiko; Hatano, Ken-ichi

2013-01-01

STK_08120 is a member of the thermoacidophile-specific DUF3211 protein family from Sulfolobus tokodaii strain 7. Its molecular function remains obscure, and sequence similarities for obtaining functional remarks are not available. In this study, the crystal structure of STK_08120 was determined at 1.79-Å resolution to predict its probable function using structure similarity searches. The structure adopts an α/β structure of a helix-grip fold, which is found in the START domain proteins with cavities for hydrophobic substrates or ligands. The detailed structural features implied that fatty acids are the primary ligand candidates for STK_08120, and binding assays revealed that the protein bound long-chain saturated fatty acids (>C14) and their trans-unsaturated types with an affinity equal to that for major fatty acid binding proteins in mammals and plants. Moreover, the structure of an STK_08120-myristic acid complex revealed a unique binding mode among fatty acid binding proteins. These results suggest that the thermoacidophile-specific protein family DUF3211 functions as a fatty acid carrier with a novel binding mode. PMID:23836863

The binding properties of cycloxaprid on insect native nAChRs partially explain the low cross-resistance with imidacloprid in Nilaparvata lugens.

PubMed

Zhang, Yixi; Xu, Xiaoyong; Bao, Haibo; Shao, Xusheng; Li, Zhong; Liu, Zewen

2018-06-06

Neonicotinoids, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) to control Nilaparvata lugens, a major rice insect pest. High imidacloprid resistance has been reported in N. lugens in laboratory and in fields. Cycloxaprid, an oxabridged cis-nitromethylene neonicotinoid, showed high insecticidal activity against N. lugens and low cross-resistance in the imidacloprid resistant strains and field populations. Binding studies have demonstrated that imidacloprid had two binding sites with different affinities (Kd = 3.18 ± 0.43 pM and 1.78 ± 0.19 nM) in N. lugens nAChRs. Cycloxaprid was poor at displacing [ 3 H]imidacloprid at its high-affinity binding site (Ki = 159.38±20.43 nM), but quite efficient at the low-affinity binding site (Ki = 1.27±0.35 nM). These data showed that cycloxaprid had overlapping binding sites with imidacloprid only at its low-affinity binding site. Therefore, the low displacement ability of cycloxaprid against imidacloprid binding at its high affinity site could partially explain the low cross-resistance of cycloxaprid in the imidacloprid resistant populations. The high insecticidal activity, low cross-resistance and different binding properties on insect nAChRs of cycloxaprid demonstrating it a potential insecticide to control N. lugens and related insect pests, especially the ones with high resistance to neonicotinoids. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Large scale affinity calculations of cyclodextrin host-guest complexes: Understanding the role of reorganization in the molecular recognition process

PubMed Central

Wickstrom, Lauren; He, Peng; Gallicchio, Emilio; Levy, Ronald M.

2013-01-01

Host-guest inclusion complexes are useful models for understanding the structural and energetic aspects of molecular recognition. Due to their small size relative to much larger protein-ligand complexes, converged results can be obtained rapidly for these systems thus offering the opportunity to more reliably study fundamental aspects of the thermodynamics of binding. In this work, we have performed a large scale binding affinity survey of 57 β-cyclodextrin (CD) host guest systems using the binding energy distribution analysis method (BEDAM) with implicit solvation (OPLS-AA/AGBNP2). Converged estimates of the standard binding free energies are obtained for these systems by employing techniques such as parallel Hamitionian replica exchange molecular dynamics, conformational reservoirs and multistate free energy estimators. Good agreement with experimental measurements is obtained in terms of both numerical accuracy and affinity rankings. Overall, average effective binding energies reproduce affinity rank ordering better than the calculated binding affinities, even though calculated binding free energies, which account for effects such as conformational strain and entropy loss upon binding, provide lower root mean square errors when compared to measurements. Interestingly, we find that binding free energies are superior rank order predictors for a large subset containing the most flexible guests. The results indicate that, while challenging, accurate modeling of reorganization effects can lead to ligand design models of superior predictive power for rank ordering relative to models based only on ligand-receptor interaction energies. PMID:25147485

Pharmacologically relevant receptor binding characteristics and 5alpha-reductase inhibitory activity of free Fatty acids contained in saw palmetto extract.

PubMed

Abe, Masayuki; Ito, Yoshihiko; Oyunzul, Luvsandorj; Oki-Fujino, Tomomi; Yamada, Shizuo

2009-04-01

Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (alpha(1)-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [(3)H]prazosin binding in rat brain in a concentration-dependent manner with IC(50) values of 23.8 to 136 microg/ml, and specific (+)-[(3)H]PN 200-110 binding with IC(50) values of 24.5 to 79.5 microg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [(3)H]N-methylscopolamine ([(3)H]NMS) binding in rat brain with IC(50) values of 56.4 to 169 microg/ml. Palmitic acid had no effect on specific [(3)H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]prazosin, [(3)H]NMS and (+)-[(3)H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to alpha(1)-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5alpha-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5alpha-reductase activity in rat liver with an IC(50) of 101 microg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5alpha-reductase activity, with IC(50) values of 42.1 to 67.6 microg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of alpha(1)-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5alpha-reductase activity.

Interplay between binding affinity and kinetics in protein-protein interactions.

PubMed

Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

2016-07-01

To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Correlation of Local Effects of DNA Sequence and Position of Beta-Alanine Inserts with Polyamide-DNA Complex Binding Affinities and Kinetics

PubMed Central

Wang, Shuo; Nanjunda, Rupesh; Aston, Karl; Bashkin, James K.; Wilson, W. David

2012-01-01

In order to better understand the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analog have been investigated with their cognate DNA sequence, 5′-TGGCTT-3′. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV-melting, biosensor-surface plasmon resonance, isothermal titration calorimetry, circular dichroism and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs, and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔCp, and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in PA binding to the mutant DNAs further confirm the position-dependent effects on PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T•A versus A•T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence dependent binding of PAs to DNA. PMID:23167504

The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

PubMed

Jaffrey, S R; Haile, D J; Klausner, R D; Harford, J B

1993-09-25

To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

PubMed Central

Julian, Mark C.; Li, Lijuan; Garde, Shekhar; Wilen, Rebecca; Tessier, Peter M.

2017-01-01

The ability of antibodies to accumulate affinity-enhancing mutations in their complementarity-determining regions (CDRs) without compromising thermodynamic stability is critical to their natural function. However, it is unclear if affinity mutations in the hypervariable CDRs generally impact antibody stability and to what extent additional compensatory mutations are required to maintain stability during affinity maturation. Here we have experimentally and computationally evaluated the functional contributions of mutations acquired by a human variable (VH) domain that was evolved using strong selections for enhanced stability and affinity for the Alzheimer’s Aβ42 peptide. Interestingly, half of the key affinity mutations in the CDRs were destabilizing. Moreover, the destabilizing effects of these mutations were compensated for by a subset of the affinity mutations that were also stabilizing. Our findings demonstrate that the accumulation of both affinity and stability mutations is necessary to maintain thermodynamic stability during extensive mutagenesis and affinity maturation in vitro, which is similar to findings for natural antibodies that are subjected to somatic hypermutation in vivo. These findings for diverse antibodies and antibody fragments specific for unrelated antigens suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability. PMID:28349921

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tiberi, M.; Magnan, J.

The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid (3H)D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid (3H)D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid (3H)U69593 (Kd = 3.31 nM, Rmore » = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by (3H)U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan (3H)ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, (3H)ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of (3H)ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with (3H)U69593. Saturation studies using the nonselective opioid (3H)bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue).« less

Methamphetamine and 3,4-methylenedioxymethamphetamine interact with central nicotinic receptors and induce their up-regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Rates, Sara; Camarasa, Jordi; Escubedo, Elena

2007-09-15

Previous work from our group indicated that {alpha}7 nicotinic acetylcholine receptors ({alpha}7 nAChR) potentially play a role in methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) neurotoxicity. The aims of the present study were two-fold: (1) to demonstrate the interaction of METH and MDMA with homomeric {alpha}7 nAChR ([{sup 3}H]methyllycaconitine binding) and other heteromeric subtypes ([{sup 3}H]epibatidine binding); and (2) to show the effects of amphetamine derivative pretreatment on the density of binding sites. METH and MDMA displaced [{sup 3}H]methyllycaconitine and [{sup 3}H]epibatidine binding in membranes from NGF-differentiated PC 12 cells and mouse brain, with K{sub i} values in the micromolar range, MDMAmore » revealing a greater affinity than METH. In addition, METH and MDMA induced a time- and concentration-dependent increase in [{sup 3}H]methyllycaconitine and [{sup 3}H]epibatidine binding; which had already been apparent after 6 h of pretreatment, and which peaked in differentiated PC 12 cells after 48 h. The highest increases were found in [{sup 3}H]epibatidine binding, with MDMA inducing higher increases than METH. Treatment with METH and MDMA increased B{sub max} of high-affinity sites for both radioligands without affecting K{sub d}. The heightened binding was inhibited by pretreatment with cycloheximide, suggesting the participation of newly synthesised proteins while inhibition of protein trafficking to plasma membrane did not block up-regulation. The effects of protein kinase and cyclophilin inhibitors on such up-regulation were explored, revealing a rapid, differential and complex regulation, similar to that described for nicotinic ligands. All of these results demonstrate that METH and MDMA have affinity for, and can interact with, nAChR, inducing their up-regulation, specially when higher doses are used. Such effects may have a role in METH- and MDMA-induced neurotoxicity, cholinergic neurotransmission, and in processes related to addiction and dependence.« less

Quantitative cumulative biodistribution of antibodies in mice

PubMed Central

Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

2014-01-01

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn’s role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn’s role in antibody PK and catabolism at the tissue level. PMID:24572100

Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria*

PubMed Central

Anashkin, Viktor A.; Salminen, Anu; Tuominen, Heidi K.; Orlov, Victor N.; Lahti, Reijo; Baykov, Alexander A.

2015-01-01

Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P1,Pn-diadenosine 5′-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ∼10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria. PMID:26400082

The use of one-bead one-compound combinatorial library technology to discover high-affinity αvβ3 integrin and cancer targeting RGD ligands with a build-in handle

PubMed Central

Xiao, Wenwu; Wang, Yan; Lau, Edmond Y.; Luo, Juntao; Yao, Nianhuan; Shi, Changying; Meza, Leah; Tseng, Harry; Maeda, Yoshiko; Kumaresan, Pappanaicken; Liu, Ruiwu; Lightstone, Felice C.; Takada, Yoshikazu; Lam, Kit S.

2012-01-01

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiological processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted-therapy and cancer imaging. In this report, one-bead-one-compound (OBOC) combinatorial libraries containing the RGD motif were designed and screened against K562 myeloid leukemia cells that had been transfected with human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a build-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68±0.08 μM) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2 to 8 fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with biotinylated-ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake. PMID:20858725

Engineering cofactor and ligand binding in an artificial neuroglobin

NASA Astrophysics Data System (ADS)

Zhang, Lei

HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a large increase in water penetration into the protein core. The inability of the mutant protein may thus either be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors.

Amyloid tracers detect multiple binding sites in Alzheimer's disease brain tissue.

PubMed

Ni, Ruiqing; Gillberg, Per-Göran; Bergfors, Assar; Marutle, Amelia; Nordberg, Agneta

2013-07-01

Imaging fibrillar amyloid-β deposition in the human brain in vivo by positron emission tomography has improved our understanding of the time course of amyloid-β pathology in Alzheimer's disease. The most widely used amyloid-β imaging tracer so far is (11)C-Pittsburgh compound B, a thioflavin derivative but other (11)C- and (18)F-labelled amyloid-β tracers have been studied in patients with Alzheimer's disease and cognitively normal control subjects. However, it has not yet been established whether different amyloid tracers bind to identical sites on amyloid-β fibrils, offering the same ability to detect the regional amyloid-β burden in the brains. In this study, we characterized (3)H-Pittsburgh compound B binding in autopsied brain regions from 23 patients with Alzheimer's disease and 20 control subjects (aged 50 to 88 years). The binding properties of the amyloid tracers FDDNP, AV-45, AV-1 and BF-227 were also compared with those of (3)H-Pittsburgh compound B in the frontal cortices of patients with Alzheimer's disease. Saturation binding studies revealed the presence of high- and low-affinity (3)H-Pittsburgh compound B binding sites in the frontal cortex (K(d1): 3.5 ± 1.6 nM; K(d2): 133 ± 30 nM) and hippocampus (K(d1):5.6 ± 2.2 nM; K(d2): 181 ± 132 nM) of Alzheimer's disease brains. The relative proportion of high-affinity to low-affinity sites was 6:1 in the frontal cortex and 3:1 in the hippocampus. One control showed both high- and low-affinity (3)H-Pittsburgh compound B binding sites (K(d1): 1.6 nM; K(d2): 330 nM) in the cortex while the others only had a low-affinity site (K(d2): 191 ± 70 nM). (3)H-Pittsburgh compound B binding in Alzheimer's disease brains was higher in the frontal and parietal cortices than in the caudate nucleus and hippocampus, and negligible in the cerebellum. Competitive binding studies with (3)H-Pittsburgh compound B in the frontal cortices of Alzheimer's disease brains revealed high- and low-affinity binding sites for BTA-1 (Ki: 0.2 nM, 70 nM), florbetapir (1.8 nM, 53 nM) and florbetaben (1.0 nM, 65 nM). BF-227 displaced 83% of (3)H-Pittsburgh compound B binding, mainly at a low-affinity site (311 nM), whereas FDDNP only partly displaced (40%). We propose a multiple binding site model for the amyloid tracers (binding sites 1, 2 and 3), where AV-45 (florbetapir), AV-1 (florbetaben), and Pittsburgh compound B, all show nanomolar affinity for the high-affinity site (binding site 1), as visualized by positron emission tomography. BF-227 shows mainly binding to site 3 and FDDNP shows only some binding to site 2. Different amyloid tracers may provide new insight into the pathophysiological mechanisms in the progression of Alzheimer's disease.

Characteristics of recombinantly expressed rat and human histamine H3 receptors.

PubMed

Wulff, Birgitte S; Hastrup, Sven; Rimvall, Karin

2002-10-18

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor. Copyright 2002 Elsevier Science B.V.

The exchanged EF-hands in calmodulin and troponin C chimeras impair the Ca²⁺-induced hydrophobicity and alter the interaction with Orai1: a spectroscopic, thermodynamic and kinetic study.

PubMed

Jensen, Drake; Reynolds, Nicole; Yang, Ya-Ping; Shakya, Shubha; Wang, Zhi-Qiang; Stuehr, Dennis J; Wei, Chin-Chuan

2015-02-15

Calmodulin (CaM) plays an important role in Ca(2+)-dependent signal transduction. Ca(2+) binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca(2+)-dependent inactivation process in store-operated Ca(2+) entry, by interacting Orai1. To understand the relationship between Ca(2+)-induced hydrophobicity and CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) are used as an informative probe to better understand the functionality of each EF-hand. ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca(2+) binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = -5.02 ± 0.13 kcal/mol) and binding affinity (K(a) = 8.92 ± 1.03 × 10(5) M(-1)). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ΔH than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s(-1) by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76 indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged. Using ANS dye to assess induced hydrophobicity showed that exchanging EFs for all Ca(2+)-bound chimeras impaired ANS fluorescence and/or binding affinity, consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai-CMBD. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the already published crystal structure.

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

PubMed

Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

1999-02-05

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

On the binding affinity of macromolecular interactions: daring to ask why proteins interact

PubMed Central

Kastritis, Panagiotis L.; Bonvin, Alexandre M. J. J.

2013-01-01

Interactions between proteins are orchestrated in a precise and time-dependent manner, underlying cellular function. The binding affinity, defined as the strength of these interactions, is translated into physico-chemical terms in the dissociation constant (Kd), the latter being an experimental measure that determines whether an interaction will be formed in solution or not. Predicting binding affinity from structural models has been a matter of active research for more than 40 years because of its fundamental role in drug development. However, all available approaches are incapable of predicting the binding affinity of protein–protein complexes from coordinates alone. Here, we examine both theoretical and experimental limitations that complicate the derivation of structure–affinity relationships. Most work so far has concentrated on binary interactions. Systems of increased complexity are far from being understood. The main physico-chemical measure that relates to binding affinity is the buried surface area, but it does not hold for flexible complexes. For the latter, there must be a significant entropic contribution that will have to be approximated in the future. We foresee that any theoretical modelling of these interactions will have to follow an integrative approach considering the biology, chemistry and physics that underlie protein–protein recognition. PMID:23235262

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.

PubMed

Pai, Sudipta; Das, Mili; Banerjee, Rahul; Dasgupta, Dipak

2011-08-01

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.

Affinity and Efficacy Studies of Tetrahydrocannabinolic Acid A at Cannabinoid Receptor Types One and Two.

PubMed

McPartland, John M; MacDonald, Christa; Young, Michelle; Grant, Phillip S; Furkert, Daniel P; Glass, Michelle

2017-01-01

Introduction: Cannabis biosynthesizes Δ 9 -tetrahydrocannabinolic acid (THCA-A), which decarboxylates into Δ 9 -tetrahydrocannabinol (THC). There is growing interest in the therapeutic use of THCA-A, but its clinical application may be hampered by instability. THCA-A lacks cannabimimetic effects; we hypothesize that it has little binding affinity at cannabinoid receptor 1 (CB 1 ). Materials and Methods: Purity of certified reference standards were tested with high performance liquid chromatography (HPLC). Binding affinity of THCA-A and THC at human (h) CB 1 and hCB 2 was measured in competition binding assays, using transfected HEK cells and [ 3 H]CP55,940. Efficacy at hCB 1 and hCB 2 was measured in a cyclic adenosine monophosphase (cAMP) assay, using a Bioluminescence Resonance Energy Transfer (BRET) biosensor. Results: The THCA-A reagent contained 2% THC. THCA-A displayed small but measurable binding at both hCB 1 and hCB 2 , equating to approximate K i values of 3.1μM and 12.5μM, respectively. THC showed 62-fold greater affinity at hCB 1 and 125-fold greater affinity at hCB 2 . In efficacy tests, THCA-A (10μM) slightly inhibited forskolin-stimulated cAMP at hCB 1 , suggestive of weak agonist activity, and no measurable efficacy at hCB 2 . Discussion: The presence of THC in our THCA-A certified standard agrees with decarboxylation kinetics (literature reviewed herein), which indicate contamination with THC is nearly unavoidable. THCA-A binding at 10μM approximated THC binding at 200nM. We therefore suspect some of our THCA-A binding curve was artifact-from its inevitable decarboxylation into THC-and the binding affinity of THCA-A is even weaker than our estimated values. We conclude that THCA-A has little affinity or efficacy at CB 1 or CB 2 .

Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

EPA Science Inventory

The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

PubMed Central

Honey, Denise M.; Best, Annie; Qiu, Huawei

2018-01-01

ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

PubMed

Yandell, C A; Dunbar, A J; Wheldrake, J F; Upton, Z

1999-09-17

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Molecular recognition of PTS-1 cargo proteins by Pex5p: implications for protein mistargeting in primary hyperoxaluria.

PubMed

Mesa-Torres, Noel; Tomic, Nenad; Albert, Armando; Salido, Eduardo; Pey, Angel L

2015-02-13

Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3-4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

Binding of RNA by the Nucleoproteins of Influenza Viruses A and B

PubMed Central

Labaronne, Alice; Swale, Christopher; Monod, Alexandre; Schoehn, Guy; Crépin, Thibaut; Ruigrok, Rob W. H.

2016-01-01

This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)6-FAM3′ RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses. PMID:27649229

Protein profile of Lupinus texensis phloem sap exudates: searching for Fe- and Zn-containing proteins.

PubMed

Lattanzio, Giuseppe; Andaluz, Sofía; Matros, Andrea; Calvete, Juan José; Kehr, Julia; Abadía, Anunciación; Abadía, Javier; López-Millán, Ana-Flor

2013-08-01

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI-MS and ESI-MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19-21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe-containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe-binding proteins in phloem sap: a metallothionein-like protein type 2B identified in the Fe-affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem-specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn-binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

GRP78 enabled micelle-based glioma targeted drug delivery.

PubMed

Ran, Danni; Mao, Jiani; Shen, Qing; Xie, Cao; Zhan, Changyou; Wang, Ruifeng; Lu, Weiyue

2017-06-10

GRP78, a specific cancer cell-surface marker, is implicated in cancer cells proliferation, apoptosis resistance, metastasis and drug resistance. l-VAP (SNTRVAP) is a tumor homing peptide exhibiting high binding affinity in vitro to GRP78 protein overexpressed on glioma, glioma stem cells, vasculogenic mimicry and neovasculature. Even though short peptides are often non-immunogenic and demonstrate high affinity to tumor cells, their targeting efficacy is always undermined by rapid blood clearance and enzymatic degradation. In the present study, two d peptides RI-VAP (retro inverso isomer of l-VAP) and d-VAP (retro isomer of l-VAP) were developed by structure-guided peptide design and retro-inverso isomerization technique for glioma targeting. RI-VAP and d-VAP were predicted to bind their receptor GRP78 protein with similar binding affinity, which was experimentally confirmed. The results of in vivo imaging demonstrated that RI-VAP and d-VAP had remarkably advantage over l-VAP for tumor accumulation. In addition, RI-VAP and d-VAP modified paclitaxel-loaded polymeric micelle had better anti-tumor efficacy in comparison to taxol, paclitaxel-loaded plain micelles and l-VAP modified micelles. Overall, the VAP modified micelles suggested in the present study could effectively achieve glioma-targeted drug delivery, validating the potential of the stable VAP peptides in improving the therapeutic efficacy of paclitaxel for glioma. Copyright © 2017 Elsevier B.V. All rights reserved.

Kir6.2-dependent high-affinity repaglinide binding to β-cell KATP channels

PubMed Central

Hansen, Ann Maria K; Hansen, John Bondo; Carr, Richard D; Ashcroft, Frances M; Wahl, Philip

2005-01-01

The β-cell KATP channel is composed of two types of subunit – the inward rectifier K+ channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2ΔN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2ΔN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37°C. Repaglinide was found to bind with low affinity (KD=59±16 nM) to SUR1 alone, but with high affinity (increased ∼150-fold) when SUR1 was co-expressed with Kir6.2 (KD=0.42±0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. Repaglinide bound with low affinity (KD=51±23 nM) to SUR1 co-expressed with Kir6.2ΔN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. In whole-cell patch-clamp experiments inhibition of Kir6.2ΔN14/SUR1 currents by both repaglinide and nateglinde is abolished. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1. PMID:15678092

Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carmen Herranz, Ma; Sanchez-Navarro, Jesus-Angel; Sauri, Ana

2005-08-15

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutantsmore » and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.« less

Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement.

PubMed

Carmen Herranz, Ma; Sanchez-Navarro, Jesús-Angel; Saurí, Ana; Mingarro, Ismael; Pallás, Vicente

2005-08-15

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.

Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Amato, R.J.; Largent, B.L.; Snowman, A.M.

1987-07-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine bindingmore » reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.« less

Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

PubMed Central

Galka, Marek M.; Rajagopalan, Nandhakishore; Buhrow, Leann M.; Nelson, Ken M.; Switala, Jacek; Cutler, Adrian J.; Palmer, David R. J.; Loewen, Peter C.; Abrams, Suzanne R.; Loewen, Michele C.

2015-01-01

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation. PMID:26197050

Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

PubMed

Galka, Marek M; Rajagopalan, Nandhakishore; Buhrow, Leann M; Nelson, Ken M; Switala, Jacek; Cutler, Adrian J; Palmer, David R J; Loewen, Peter C; Abrams, Suzanne R; Loewen, Michele C

2015-01-01

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.

2011-02-01

Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17{beta} to the four rainbow trout ER isoformsmore » with that of three known environmental estrogens 17{alpha}-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ER{alpha} subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17{beta}, bisphenol A binds less strongly to all four receptors, 17{alpha}-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the {alpha} subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.« less

Use of 2-(/sup 125/I)iodomelatonin to characterize melatonin binding sites in chicken retina

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dubocovich, M.L.; Takahashi, J.S.

2-(/sup 125/I)Iodomelatonin binds with high affinity to a site possessing the pharmacological characteristics of a melatonin receptor in chicken retinal membranes. The specific binding of 2-(/sup 125/I)iodomelatonin is stable, saturable, and reversible. Saturation experiments indicated that 2-(/sup 125/I)iodomelatonin labeled a single class of sites with an affinity constant (Kd) of 434 +/- 56 pM and a total number of binding sites (Bmax) of 74.0 +/- 13.6 fmol/mg of protein. The affinity constant obtained from kinetic analysis was in close agreement with that obtained in saturation experiments. Competition experiments showed a monophasic reduction of 2-(/sup 125/I)iodomelatonin binding with a pharmacological ordermore » of indole amine affinities characteristic of a melatonin receptor: 2-iodomelatonin greater than 6-chloromelatonin greater than or equal to melatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 6-hydroxymelatonin greater than or equal to 6-methoxymelatonin much greater than N-acetyltryptamine greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine greater than 5-hydroxytryptamine (inactive). The affinities of these melatonin analogs in competing for 2-(/sup 125/I)iodomelatonin binding sites were correlated closely with their potencies for inhibition of the calcium-dependent release of (3H)dopamine from chicken and rabbit retinas, indicating association of the binding site with a functional response regulated by melatonin. The results indicate that 2-(/sup 125/I)iodomelatonin is a selective, high-affinity radioligand for the identification and characterization of melatonin receptor sites.« less

Computational Design of Ligand Binding Proteins with High Affinity and Selectivity

PubMed Central

Dou, Jiayi; Doyle, Lindsey; Nelson, Jorgen W.; Schena, Alberto; Jankowski, Wojciech; Kalodimos, Charalampos G.; Johnsson, Kai; Stoddard, Barry L.; Baker, David

2014-01-01

The ability to design proteins with high affinity and selectivity for any given small molecule would have numerous applications in biosensing, diagnostics, and therapeutics, and is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition phenomena. Attempts to design ligand binding proteins have met with little success, however, and the computational design of precise molecular recognition between proteins and small molecules remains an “unsolved problem”1. We describe a general method for the computational design of small molecule binding sites with pre-organized hydrogen bonding and hydrophobic interfaces and high overall shape complementary to the ligand, and use it to design protein binding sites for the steroid digoxigenin (DIG). Of 17 designs that were experimentally characterized, two bind DIG; the highest affinity design has the lowest predicted interaction energy and the most pre-organized binding site in the set. A comprehensive binding-fitness landscape of this design generated by library selection and deep sequencing was used to guide optimization of binding affinity to a picomolar level, and two X-ray co-crystal structures of optimized complexes show atomic level agreement with the design models. The designed binder has a high selectivity for DIG over the related steroids digitoxigenin, progesterone, and β-estradiol, which can be reprogrammed through the designed hydrogen-bonding interactions. Taken together, the binding fitness landscape, co-crystal structures, and thermodynamic binding parameters illustrate how increases in binding affinity can result from distal sequence changes that limit the protein ensemble to conformers making the most energetically favorable interactions with the ligand. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics, and diagnostics. PMID:24005320

Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering

PubMed Central

Thakkar, Shraddha; Nanaware-Kharade, Nisha; Celikel, Reha; Peterson, Eric C.; Varughese, Kottayil I.

2014-01-01

Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP. PMID:24419156

Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells.

PubMed

Montgomery, M D; Bylund, D B

2010-02-01

The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

Replication protein A 32 interacts through a similar binding interface with TIPIN, XPA, and UNG2.

PubMed

Ali, Seikh Imtiaz; Shin, Jae-Sun; Bae, Sung-Hun; Kim, Byoungkook; Choi, Byong-Seok

2010-07-01

The 32kDa subunit of replication protein A (RPA32) is involved in various DNA repair systems such as nucleotide excision repair, base excision repair, and homologous recombination. In these processes, RPA32 interacts with different binding partners via its C-terminal domain (RPA32C; residues 172-270). It has been reported recently that RPA32C also interacts with TIPIN during the intra-S checkpoint. To determine the significance of the interaction of RPA32C with TIPIN, we have examined the interaction mode using NMR spectroscopy and an in silico modeling approach. Here, we show that TIPIN(185-218), which shares high sequence similarity with XPA(10-43) and UNG2(56-89), is less ordered in the free state and then forms a longer alpha-helix upon binding to RPA32C. The binding interface between TIPIN(185-218) and RPA32C is similar to those of XPA and UNG2, but its mode of interaction is different. The results suggest that RPA32 is an exchange point for multiple proteins involved in DNA repair, homologous recombination, and checkpoint processes and that it binds to different partners with comparable binding affinity using a single site. Copyright 2010 Elsevier Ltd. All rights reserved.

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

PubMed

Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

2016-09-01

Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion. © The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Evidence for a G protein-coupled diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) receptor binding site in lung membranes from rat.

PubMed

Laubinger, W; Reiser, G

1999-01-29

Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [3H]diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [3H]Ap4A with similar affinity for other diadenosine polyphosphates ApnA (n = 2 to 6). Displacement studies with different nucleotides revealed that the [3H]Ap4A binding site was different from P2X and P2Y2 receptor binding sites. Pretreatment of lung membranes with GTPgammaS or GTP in the presence of Mg2+ increased the Ki for Ap4A from 91 nM to 5.1 microM, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [35S]GTPgammaS binding (to Galpha proteins) to lung membranes by Ap4A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap4A binding site in lung membranes.

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

PubMed Central

Chess-Williams, R.; Chapple, C. R.; Verfurth, F.; Noble, A. J.; Couldwell, C. J.; Michel, M. C.

1996-01-01

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means. PMID:8937710

Exploring the free-energy landscape of carbohydrate-protein complexes: development and validation of scoring functions considering the binding-site topology

NASA Astrophysics Data System (ADS)

Eid, Sameh; Saleh, Noureldin; Zalewski, Adam; Vedani, Angelo

2014-12-01

Carbohydrates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the structure-based design of carbohydrate-based ligands. We assembled a diverse data set comprising 273 carbohydrate-protein crystal structures with known binding affinity and evaluated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as combinations thereof. Unfortunately, the tested functions were not capable of reproducing binding affinities in the studied complexes. To simplify the complex free-energy surface of carbohydrate-protein systems, we classified the studied proteins according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classification scheme reproduced binding affinities in the carbohydrate data set with an r 2 of 0.71 and root-mean-squared-error of 1.25 kcal/mol ( N = 236). The improvement in model performance underlines the significance of the differences in the local micro-environments of carbohydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and solvent exposure.

DFT-based ranking of zinc-binding groups in histone deacetylase inhibitors.

PubMed

Vanommeslaeghe, K; Loverix, S; Geerlings, P; Tourwé, D

2005-11-01

Histone deacetylases (HDACs) have recently attracted considerable interest as targets in the treatment of cell proliferative diseases such as cancer. In the present work, a general framework is proposed for chemical groups that bind into the HDAC catalytic core. Based on this framework, a series of groups was selected for further investigation. A method was developed to rank the HDAC inhibitory potential of these moieties at the B3LYP/6-31G* level, making use of extra diffuse functions and of the PCM solvation model where appropriate. The resulting binding geometries indicate that very stringent constraints should be satisfied in order to have bidental zinc chelation, and even more so to have a strong binding affinity, which makes it difficult to predict the binding mode and affinity of such zinc-binding groups. The chemical hardness and the pK(a) were identified as important criteria for the binding affinity. Also, the hydrophilicity may have a direct influence on the binding affinity. The calculated binding energies were qualitatively validated with experimental results from the literature, and were shown to be meaningful for the purpose of ranking. Additionally, the insights gained from the present work may be useful for increasing the accuracy of QSAR models by providing a rational basis for selecting descriptors.

Receptor interaction profiles of novel psychoactive tryptamines compared with classic hallucinogens.

PubMed

Rickli, Anna; Moning, Olivier D; Hoener, Marius C; Liechti, Matthias E

2016-08-01

The present study investigated interactions between the novel psychoactive tryptamines DiPT, 4-OH-DiPT, 4-OH-MET, 5-MeO-AMT, and 5-MeO-MiPT at monoamine receptors and transporters compared with the classic hallucinogens lysergic acid diethylamide (LSD), psilocin, N,N-dimethyltryptamine (DMT), and mescaline. We investigated binding affinities at human monoamine receptors and determined functional serotonin (5-hydroxytryptamine [5-HT]) 5-HT2A and 5-HT2B receptor activation. Binding at and the inhibition of human monoamine uptake transporters and transporter-mediated monoamine release were also determined. All of the novel tryptamines interacted with 5-HT2A receptors and were partial or full 5-HT2A agonists. Binding affinity to the 5-HT2A receptor was lower for all of the tryptamines, including psilocin and DMT, compared with LSD and correlated with the reported psychoactive doses in humans. Several tryptamines, including psilocin, DMT, DiPT, 4-OH-DiPT, and 4-OH-MET, interacted with the serotonin transporter and partially the norepinephrine transporter, similar to 3,4-methylenedioxymethamphetamine but in contrast to LSD and mescaline. LSD but not the tryptamines interacted with adrenergic and dopaminergic receptors. In conclusion, the receptor interaction profiles of the tryptamines predict hallucinogenic effects that are similar to classic serotonergic hallucinogens but also MDMA-like psychoactive properties. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition

PubMed Central

Stieglitz, Kimberly A.; Pastra-Landis, Styliani C.; Xia, Jiarong; Tsuruta, Hiro; Kantrowitz, Evan R.

2005-01-01

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low activity low affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase. PMID:15890205

A dye-binding assay for measurement of the binding of Cu(II) to proteins.

PubMed

Wilkinson-White, Lorna E; Easterbrook-Smith, Simon B

2008-10-01

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).

Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant.

PubMed

Mumford, A D; Nisar, S; Darnige, L; Jones, M L; Bachelot-Loza, C; Gandrille, S; Zinzindohoue, F; Fischer, A-M; Mundell, S J; Gaussem, P

2013-03-01

Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding. © 2012 International Society on Thrombosis and Haemostasis.

Metal loading levels influence on REE distribution on humic acid: Experimental and Modelling approach

NASA Astrophysics Data System (ADS)

Marsac, R.; Davranche, M.; Gruau, G.; Dia, A.

2009-04-01

In natural organic-rich waters, rare earth elements (REE) speciation is mainly controlled by organic colloids such as humic acid (HA). Different series of REE-HA complexation experiments performed at several metal loading (REE/C) displayed two pattern shapes (i) at high metal loading, a middle-REE (MREE) downward concavity, and (ii) at low metal loading, a regular increase from La to Lu (e.g. Sonke and Salters, 2006; Pourret et al., 2007). Both REE patterns might be related to REE binding with different surface sites on HA. To understand REE-HA binding, REE-HA complexation experiments at various metals loading were carried out using ultrafiltration combined with ICP-MS measurements, for the 14 REE simultaneously. The patterns of the apparent coefficients of REE partition between HA and the inorganic solution (log Kd) evolved regularly according to the metal loading. The REE patterns presented a MREE downward concavity at low loading and a regular increase from La to Lu at high loading. The dataset was modelled with Model VI by adjusting two specific parameters, log KMA, the apparent complexation constant of HA low affinity sites and DLK2, the parameter increasing high affinity sites binding strength. Experiments and modelling provided evidence that HA high affinity sites controlled the REE binding with HA at low metal loading. The REE-HA complex could be as multidentate complexes with carboxylic or phenolic sites or potentially with sites constituted of N, P or S as donor atoms. Moreover, these high affinity sites could be different for light and heavy REE, because heavy REE have higher affinity for these sites, in low density, and could saturate them. These new Model VI parameter sets allowed the prediction of the REE-HA pattern shape evolution on a large range of pH and metal loading. According to the metal loading, the evolution of the calculated REE patterns was similar to the various REE pattern observed in natural acidic organic-rich waters (pH<7 and DOC>10 mg L-1). As a consequence, the metal loading could be the key parameter controlling the REE pattern in organic-rich waters.

Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy.

PubMed

Orwig, Kevin S; Lassetter, McKensie R; Hadden, M Kyle; Dix, Thomas A

2009-04-09

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

PubMed Central

2015-01-01

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. PMID:25073654

Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges

PubMed Central

Naiser, Thomas; Ehler, Oliver; Kayser, Jona; Mai, Timo; Michel, Wolfgang; Ott, Albrecht

2008-01-01

Background The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations. Results We performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form). Conclusion We have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role. PMID:18477387

Synthesis of (nor)tropeine (di)esters and allosteric modulation of glycine receptor binding.

PubMed

Maksay, Gábor; Nemes, Péter; Vincze, Zoltán; Bíró, Timea

2008-02-15

(Hetero)aromatic mono- and diesters of tropine and nortropine were prepared. Modulation of [3H]strychnine binding to glycine receptors of rat spinal cord was examined with a ternary allosteric model. The esters displaced [3H]strychnine binding with nano- or micromolar potencies and strong negative cooperativity. Coplanarity and distance of the ester moieties of diesters affected the binding affinity being nanomolar for isophthaloyl-bistropane and nortropeines. Nortropisetron had the highest affinity (K(A) approximately 10 nM). Two esters displayed negative cooperativity with glycine in displacement, while three esters of low-affinity and nortropisetron exerted positive cooperativity with glycine.

The C Terminus of Formin FMNL3 Accelerates Actin Polymerization and Contains a WH2 Domain-like Sequence That Binds Both Monomers and Filament Barbed Ends*

PubMed Central

Heimsath, Ernest G.; Higgs, Henry N.

2012-01-01

Formin proteins are actin assembly factors that accelerate filament nucleation then remain on the elongating barbed end and modulate filament elongation. The formin homology 2 (FH2) domain is central to these activities, but recent work has suggested that additional sequences enhance FH2 domain function. Here we show that the C-terminal 76 amino acids of the formin FMNL3 have a dramatic effect on the ability of the FH2 domain to accelerate actin assembly. This C-terminal region contains a WASp homology 2 (WH2)-like sequence that binds actin monomers in a manner that is competitive with other WH2 domains and with profilin. In addition, the C terminus binds filament barbed ends. As a monomer, the FMNL3 C terminus inhibits actin polymerization and slows barbed end elongation with moderate affinity. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high affinity inhibition of barbed end elongation. These properties are not common to all formin C termini, as those of mDia1 and INF2 do not behave similarly. Interestingly, mutation of two aliphatic residues, which blocks high affinity actin binding by the WH2-like sequence, has no effect on the ability of the C terminus to enhance FH2-mediated polymerization. However, mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement. These results illustrate that the C termini of formins are highly diverse in their interactions with actin. PMID:22094460

A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

PubMed

Wall, Jonathan S; Williams, Angela; Richey, Tina; Stuckey, Alan; Huang, Ying; Wooliver, Craig; Macy, Sallie; Heidel, Eric; Gupta, Neil; Lee, Angela; Rader, Brianna; Martin, Emily B; Kennel, Stephen J

2013-01-01

Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses

PubMed Central

Grünewald, Nora; Salvatico, Charlotte; Kress, Vanessa

2018-01-01

Abstract The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells. PMID:29464196

Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

PubMed

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

PubMed Central

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible. PMID:23874971

Receptor interaction profiles of novel N-2-methoxybenzyl (NBOMe) derivatives of 2,5-dimethoxy-substituted phenethylamines (2C drugs).

PubMed

Rickli, Anna; Luethi, Dino; Reinisch, Julian; Buchy, Danièle; Hoener, Marius C; Liechti, Matthias E

2015-12-01

N-2-methoxybenzyl-phenethylamines (NBOMe drugs) are newly used psychoactive substances with poorly defined pharmacological properties. The aim of the present study was to characterize the receptor binding profiles of a series of NBOMe drugs compared with their 2,5-dimethoxy-phenethylamine analogs (2C drugs) and lysergic acid diethylamide (LSD) in vitro. We investigated the binding affinities of 2C drugs (2C-B, 2C-C, 2C-D, 2C-E, 2C-H, 2C-I, 2C-N, 2C-P, 2C-T-2, 2C-T-4, 2C-T-7, and mescaline), their NBOMe analogs, and LSD at monoamine receptors and determined functional 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2B receptor activation. Binding at and the inhibition of monoamine uptake transporters were also determined. Human cells that were transfected with the respective human receptors or transporters were used (with the exception of trace amine-associated receptor-1 [TAAR1], in which rat/mouse receptors were used). All of the compounds potently interacted with serotonergic 5-HT2A, 5-HT2B, 5-HT2C receptors and rat TAAR1 (most Ki and EC50: <1 μM). The N-2-methoxybenzyl substitution of 2C drugs increased the binding affinity at serotonergic 5-HT2A, 5-HT2C, adrenergic α1, dopaminergic D1-3, and histaminergic H1 receptors and monoamine transporters but reduced binding to 5-HT1A receptors and TAAR1. As a result, NBOMe drugs were very potent 5-HT2A receptor agonists (EC50: 0.04-0.5 μM) with high 5-HT2A/5-HT1A selectivity and affinity for adrenergic α1 receptors (Ki: 0.3-0.9 μM) and TAAR1 (Ki: 0.06-2.2 μM), similar to LSD, but not dopaminergic D1-3 receptors (most Ki:>1 μM), unlike LSD. The binding profile of NBOMe drugs predicts strong hallucinogenic effects, similar to LSD, but possibly more stimulant properties because of α1 receptor interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of monocarboxylate transporter 1 (MCT1) binding affinity for Basigin gene products and L1cam.

PubMed

Howard, John; Finch, Nicole A; Ochrietor, Judith D

2010-07-01

The purpose of this study was to determine the binding affinities of Basigin gene products and neural cell adhesion molecule L1cam for monocarboxylate transporter-1 (MCT1). ELISA binding assays were performed in which recombinant proteins of the transmembrane domains of Basigin gene products and L1cam were incubated with MCT1 captured from mouse brain. It was determined that Basigin gene products bind MCT1 with moderate affinity, but L1cam does not bind MCT1. Despite a high degree of sequence conservation between Basigin gene products and L1cam, the sequences are different enough to prevent L1cam from interacting with MCT1.

A distal point mutation in the streptavidin-biotin complex preserves structure but diminishes binding affinity: experimental evidence of electronic polarization effects?

PubMed

Baugh, Loren; Le Trong, Isolde; Cerutti, David S; Gülich, Susanne; Stayton, Patrick S; Stenkamp, Ronald E; Lybrand, Terry P

2010-06-08

We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in biotin binding affinity without disrupting the equilibrium complex structure. The F130L mutation creates a small cavity occupied by a water molecule; however, all neighboring side chain positions are preserved, and protein-biotin hydrogen bonds are unperturbed. Molecular dynamics simulations reveal a reduced mobility of biotin binding residues but no observable destabilization of protein-ligand interactions. Our combined structural and computational studies suggest that the additional water molecule may affect binding affinity through an electronic polarization effect that impacts the highly cooperative hydrogen bonding network in the biotin binding pocket.

Effector analogues detect varied allosteric roles for conserved protein-effector interactions in pyruvate kinase isozymes†

PubMed Central

Alontaga, Aileen Y.; Fenton, Aron W.

2011-01-01

The binding site for allosteric inhibitor (amino acid) is highly conserved between human liver pyruvate kinase (hL-PYK) and the rabbit muscle isozyme (rM1-PYK). To detail similarities/differences in the allosteric function of these two homologs, we quantified the binding of 45 amino acid analogues to hL-PYK and their allosteric impact on affinity for the substrate, phosphoenolpyruvate (PEP). This complements a similar study previously completed for rM1-PYK. In hL-PYK, the minimum chemical requirements for effector binding are the same as those identified for rM1-PYK (i.e. the L-2-aminopropanaldehyde substructure of the effector is primarily responsible for binding). However different regions of the effector determine the magnitude of the allosteric response in hL-PYK vs. rM1-PYK. This finding is inconsistent with the idea that allosteric pathways are conserved between homologs of a protein family. PMID:21261284

Engineering vanilloid-sensitivity into the rat TRPV2 channel

PubMed Central

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-01-01

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1. DOI: http://dx.doi.org/10.7554/eLife.16409.001 PMID:27177419

Engineering vanilloid-sensitivity into the rat TRPV2 channel.

PubMed

Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres; Krepkiy, Dmitriy; Bae, Chanhyung; Pearce, Larry V; Blumberg, Peter M; Newstead, Simon; Swartz, Kenton J

2016-05-13

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1.

Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marks, J.L.; Ax, R.L.

1985-08-01

The binding of the glycosaminoglycan (3H) heparin to bull spermatozoa was compared with nonreturn rates of dairy bulls. Semen samples from five bulls above and five below an average 71% nonreturn rate were used. Samples consisted of first and second ejaculates on a single day collected 1 d/wk for up to 5 consecutive wk. Saturation binding assays using (TH) heparin were performed to quantitate the binding characteristics of each sample. Scatchard plot analyses indicated a significant difference in the binding affinity for (TH) heparin between bulls of high and low fertility. Dissociation constants were 69.0 and 119.3 pmol for bullsmore » of high and low fertility, respectively. In contrast, the number of binding sites for (TH) heparin did not differ significantly among bulls. Differences in binding affinity of (TH) heparin to bull sperm might be used to predict relative fertility of dairy bulls.« less

Enhanced Delivery of Galanin Conjugates to the Brain through Bioengineering of the Anti-Transferrin Receptor Antibody OX26.

PubMed

Thom, George; Burrell, Matthew; Haqqani, Arsalan S; Yogi, Alvaro; Lessard, Etienne; Brunette, Eric; Delaney, Christie; Baumann, Ewa; Callaghan, Deborah; Rodrigo, Natalia; Webster, Carl I; Stanimirovic, Danica B

2018-04-02

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having K D s of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

PubMed Central

Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

2001-01-01

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5.

PubMed

Roche, Jennifer Virginia; Survery, Sabeen; Kreida, Stefan; Nesverova, Veronika; Ampah-Korsah, Henry; Gourdon, Maria; Deen, Peter M T; Törnroth-Horsefield, Susanna

2017-09-01

The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser 256 , Ser 261 , Ser 264 , and Thr 269 ), of which Ser 256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

PubMed

Epps, D E; Raub, T J; Caiolfa, V; Chiari, A; Zamai, M

1999-01-01

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

PubMed Central

Pintor, J.; Torres, M.; Castro, E.; Miras-Portugal, M. T.

1991-01-01

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site. PMID:1912985

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864