Structural elucidation of the DFG-Asp in and DFG-Asp out states of TAM kinases and insight into the selectivity of their inhibitors.

PubMed

Messoussi, Abdellah; Peyronnet, Lucile; Feneyrolles, Clémence; Chevé, Gwénaël; Bougrin, Khalid; Yasri, Aziz

2014-10-10

Structural elucidation of the active (DFG-Asp in) and inactive (DFG-Asp out) states of the TAM family of receptor tyrosine kinases is required for future development of TAM inhibitors as drugs. Herein we report a computational study on each of the three TAM members Tyro-3, Axl and Mer. DFG-Asp in and DFG-Asp out homology models of each one were built based on the X-ray structure of c-Met kinase, an enzyme with a closely related sequence. Structural validation and in silico screening enabled identification of critical amino acids for ligand binding within the active site of each DFG-Asp in and DFG-Asp out model. The position and nature of amino acids that differ among Tyro-3, Axl and Mer, and the potential role of these residues in the design of selective TAM ligands, are discussed.

Identification, characterization and application of a G-quadruplex structured DNA aptamer against cancer biomarker protein anterior gradient homolog 2.

PubMed

Wu, Jie; Wang, Chi; Li, Xilan; Song, Yanling; Wang, Wei; Li, Cong; Hu, Jia; Zhu, Zhi; Li, Jiuxing; Zhang, Weiyun; Lu, Zhongxian; Yang, Chaoyong James

2012-01-01

Anterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d) values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2. In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.

Synthetic, switchable enzymes

PubMed Central

Norris, Vic; Krylov, Sergey N.; Agarwal, Pratul K.; White, Glenn J.

2017-01-01

The construction of switchable, radiation-controlled, aptameric enzymes alias swenzymes is, in principle, feasible. We propose a strategy to make such catalysts from two (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a two-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker so bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low intensity, non-ionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate, product-capturing, aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. PMID:28448969

Synthetic, Switchable Enzymes.

PubMed

Norris, Vic; Krylov, Sergey N; Agarwal, Pratul K; White, Glenn J

2017-01-01

The construction of switchable, radiation-controlled, aptameric enzymes - "swenzymes" - is, in principle, feasible. We propose a strategy to make such catalysts from 2 (or more) aptamers each selected to bind specifically to one of the substrates in, for example, a 2-substrate reaction. Construction of a combinatorial library of candidate swenzymes entails selecting a set of a million aptamers that bind one substrate and a second set of a million aptamers that bind the second substrate; the aptamers in these sets are then linked pairwise by a linker, thus bringing together the substrates. In the presence of the substrates, some linked aptamer pairs catalyze the reaction when exposed to external energy in the form of a specific frequency of low-intensity, nonionizing electromagnetic or acoustic radiation. Such swenzymes are detected via a separate product-capturing aptamer that changes conformation on capturing the product; this altered conformation allows it (1) to bind to every potential swenzyme in its vicinity (thereby giving a higher probability of capture to the swenzymes that generate the product) and (2) to bind to a sequence on a magnetic bead (thereby permitting purification of the swenzyme plus product-capturing aptamer by precipitation). Attempts to implement the swenzyme strategy may help elucidate fundamental problems in enzyme catalysis. © 2017 S. Karger AG, Basel.

Mechanistic models enable the rational use of in vitro drug-target binding kinetics for better drug effects in patients.

PubMed

de Witte, Wilhelmus E A; Wong, Yin Cheong; Nederpelt, Indira; Heitman, Laura H; Danhof, Meindert; van der Graaf, Piet H; Gilissen, Ron A H J; de Lange, Elizabeth C M

2016-01-01

Drug-target binding kinetics are major determinants of the time course of drug action for several drugs, as clearly described for the irreversible binders omeprazole and aspirin. This supports the increasing interest to incorporate newly developed high-throughput assays for drug-target binding kinetics in drug discovery. A meaningful application of in vitro drug-target binding kinetics in drug discovery requires insight into the relation between in vivo drug effect and in vitro measured drug-target binding kinetics. In this review, the authors discuss both the relation between in vitro and in vivo measured binding kinetics and the relation between in vivo binding kinetics, target occupancy and effect profiles. More scientific evidence is required for the rational selection and development of drug-candidates on the basis of in vitro estimates of drug-target binding kinetics. To elucidate the value of in vitro binding kinetics measurements, it is necessary to obtain information on system-specific properties which influence the kinetics of target occupancy and drug effect. Mathematical integration of this information enables the identification of drug-specific properties which lead to optimal target occupancy and drug effect in patients.

Invasion of host cells by malaria parasites: a tale of two protein families.

PubMed

Iyer, Jayasree; Grüner, Anne Charlotte; Rénia, Laurent; Snounou, Georges; Preiser, Peter R

2007-07-01

Malaria parasites are obligate intracellular parasites whose invasive stages select and invade the unique host cell in which they can develop with exquisite specificity and efficacy. Most studies aimed at elucidating the molecules and the mechanisms implicated in the selection and invasion processes have been conducted on the merozoite, the stage that invades erythrocytes to perpetuate the pathological cycles of parasite multiplication in the blood. Bioinformatic analysis has helped identify the members of two parasite protein families, the reticulocyte-binding protein homologues (RBL) and erythrocyte binding like (EBL), in recently sequenced genomes of different Plasmodium species. In this article we review data from classical studies and gene disruption experiments that are helping to illuminate the role of these proteins in the selection-invasion processes. The manner in which subsets of proteins from each of the families act in concert suggests a model to explain the ability of the parasites to use alternate pathways of invasion. Future perspectives and implications are discussed.

Tumor Genomic Profiling in Breast Cancer Patients Using Targeted Massively Parallel Sequencing

DTIC Science & Technology

2014-01-01

binding domain mutations in ESR1 ) were identified. Analysis is currently underway to further elucidate causes of resistance in those cases where the... ESR1 , the gene that encodes the estrogen receptor (Wagle, Garraway, and Arteaga, unpublished results). Given the potential importance of ESR...translocations in ER+ breast cancer, we have further modified our bait design to include genomic coordinates across select introns in ESR1 . In addition, two

Heterodimerization of mu and delta opioid receptors: A role in opiate synergy.

PubMed

Gomes, I; Jordan, B A; Gupta, A; Trapaidze, N; Nagy, V; Devi, L A

2000-11-15

Opiate analgesics are widely used in the treatment of severe pain. Because of their importance in therapy, different strategies have been considered for making opiates more effective while curbing their liability to be abused. Although most opiates exert their analgesic effects primarily via mu opioid receptors, a number of studies have shown that delta receptor-selective drugs can enhance their potency. The molecular basis for these findings has not been elucidated previously. In the present study, we examined whether heterodimerization of mu and delta receptors could account for the cross-modulation previously observed between these two receptors. We find that co-expression of mu and delta receptors in heterologous cells followed by selective immunoprecipitation results in the isolation of mu-delta heterodimers. Treatment of these cells with extremely low doses of certain delta-selective ligands results in a significant increase in the binding of a mu receptor agonist. Similarly, treatment with mu-selective ligands results in a significant increase in the binding of a delta receptor agonist. This robust increase is also seen in SKNSH cells that endogenously express both mu and delta receptors. Furthermore, we find that a delta receptor antagonist enhances both the potency and efficacy of the mu receptor signaling; likewise a mu antagonist enhances the potency and efficacy of the delta receptor signaling. A combination of agonists (mu and delta receptor selective) also synergistically binds and potentiates signaling by activating the mu-delta heterodimer. Taken together, these studies show that heterodimers exhibit distinct ligand binding and signaling characteristics. These findings have important clinical ramifications and may provide new foundations for more effective therapies.

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L

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

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott

2010-06-25

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the {alpha}-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the designmore » of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques - yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis - to elucidate specificity determinants for binding to Bcl-x{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub L} selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub L}-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x{sub L} binders.« less

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

Allosteric mechanism controls traffic in the chaperone/usher pathway.

PubMed

Di Yu, Xiao; Dubnovitsky, Anatoly; Pudney, Alex F; Macintyre, Sheila; Knight, Stefan D; Zavialov, Anton V

2012-11-07

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Using the QCM Biosensor-Based T7 Phage Display Combined with Bioinformatics Analysis for Target Identification of Bioactive Small Molecule.

PubMed

Takakusagi, Yoichi; Takakusagi, Kaori; Sugawara, Fumio; Sakaguchi, Kengo

2018-01-01

Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.

[Cytochemical localization and properties of selected nucleolytic enzymes].

PubMed

Sierakowska, Halina

2015-01-01

In the article there are shortly outlined studies on cytochemical localization of selected nucleolytic enzymes carried out between 1957-1986 by David Shugar and his coworkers. The histochemical localization of several nucleolytic enzymes in animal and plant tissues was determined by synthesis of specific substrates, alpha-naphthyl esters of 5'- and 3'-nucleotides and their derivatives. In rat tissues phosphodiesterase I was localized in the plasma membrane whereas phosphodiesterase II in the lizosomes, reflecting their physiological roles. The localization of pancreatic type ribonuclease in animal tissues was determined, indicating its role in extracellular digestion. Plant nucleotide pyrophosphatase was localized in several tissues, purified to near homogeneity from potato tubers and its properties and substrate specificity were determined. Application of this enzyme for removal of m7GMP from the "cap" of eukaryotic mRNA allowed to elucidate the role of "cap" in mRNA binding to ribosomes in the process of translation. Furthermore, cyclic nucleotide phosphodiesterase was isolated from potato tubers and its physicochemical properties, oligomeric structure and substrate specificity were elucidated.

Mechanosensing Potentials Gate Fuel Consumption in a Bipedal DNA Nanowalker

NASA Astrophysics Data System (ADS)

Tee, Shern Ren; Hu, Xinpeng; Loh, Iong Ying; Wang, Zhisong

2018-03-01

A bipedal DNA nanowalker was recently reported to convert chemical energy into directional motion autonomously and efficiently. To elucidate its chemomechanical coupling mechanisms, three-dimensional molecular modeling is used to obtain coarse-grained foot-track binding potentials of the DNA nanowalker via unbiased and biased sampling techniques (for the potentials' basin and high-energy edges, respectively). The binding state that is protected against fuel-induced dissociation responds asymmetrically to forward versus backward forces, unlike the unprotected state, demonstrating a mechanosensing capability to gate fuel binding. Despite complex DNA mechanics, the foot-track potential exhibits a surprisingly neat three-part profile, offering some general guidelines to rationally design efficient nanowalkers. Subsequent modeling of the bipedal walker attached to the track gives estimates of the free energy for each bipedal state, showing how the mechanosensing foot-track binding breaks the symmetry between the rear and front feet, enabling the rear foot to be selectively dissociated by fuel and generating efficient chemomechanical coupling.

MutaBind estimates and interprets the effects of sequence variants on protein-protein interactions.

PubMed

Li, Minghui; Simonetti, Franco L; Goncearenco, Alexander; Panchenko, Anna R

2016-07-08

Proteins engage in highly selective interactions with their macromolecular partners. Sequence variants that alter protein binding affinity may cause significant perturbations or complete abolishment of function, potentially leading to diseases. There exists a persistent need to develop a mechanistic understanding of impacts of variants on proteins. To address this need we introduce a new computational method MutaBind to evaluate the effects of sequence variants and disease mutations on protein interactions and calculate the quantitative changes in binding affinity. The MutaBind method uses molecular mechanics force fields, statistical potentials and fast side-chain optimization algorithms. The MutaBind server maps mutations on a structural protein complex, calculates the associated changes in binding affinity, determines the deleterious effect of a mutation, estimates the confidence of this prediction and produces a mutant structural model for download. MutaBind can be applied to a large number of problems, including determination of potential driver mutations in cancer and other diseases, elucidation of the effects of sequence variants on protein fitness in evolution and protein design. MutaBind is available at http://www.ncbi.nlm.nih.gov/projects/mutabind/. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Heterodimerization of μ and δ Opioid Receptors: A Role in Opiate Synergy

PubMed Central

Gomes, I.; Jordan, B. A.; Gupta, A.; Trapaidze, N.; Nagy, V.; Devi, L. A.

2011-01-01

Opiate analgesics are widely used in the treatment of severe pain. Because of their importance in therapy, different strategies have been considered for making opiates more effective while curbing their liability to be abused. Although most opiates exert their analgesic effects primarily via μ opioid receptors, a number of studies have shown that δ receptor-selective drugs can enhance their potency. The molecular basis for these findings has not been elucidated previously. In the present study, we examined whether heterodimerization of μ and δ receptors could account for the cross-modulation previously observed between these two receptors. We find that co-expression of μ and δ receptors in heterologous cells followed by selective immunoprecipitation results in the isolation of μ–δ heterodimers. Treatment of these cells with extremely low doses of certain δ-selective ligands results in a significant increase in the binding of a μ receptor agonist. Similarly, treatment with μ-selective ligands results in a significant increase in the binding of a δ receptor agonist. This robust increase is also seen in SKNSH cells that endogenously express both μ and δ receptors. Furthermore, we find that a δ receptor antagonist enhances both the potency and efficacy of the μ receptor signaling; likewise a μ antagonist enhances the potency and efficacy of the δ receptor signaling. A combination of agonists (μ and δ receptor selective) also synergistically binds and potentiates signaling by activating the μ–δ heterodimer. Taken together, these studies show that heterodimers exhibit distinct ligand binding and signaling characteristics. These findings have important clinical ramifications and may provide new foundations for more effective therapies. PMID:11069979

Identification of the pharmacophore of the CC chemokine-binding proteins Evasin-1 and -4 using phage display.

PubMed

Bonvin, Pauline; Dunn, Steven M; Rousseau, François; Dyer, Douglas P; Shaw, Jeffrey; Power, Christine A; Handel, Tracy M; Proudfoot, Amanda E I

2014-11-14

To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Structure Determination of Ornithine-Linked Cisplatin by Infrared Multiple Photon Dissociation Action Spectroscopy

NASA Astrophysics Data System (ADS)

He, Chenchen; Kimutai, Bett; Hamlow, Lucas; Roy, Harrison; Nei, Y.-W.; Bao, Xun; Gao, Juehan; Martens, Jonathan K.; Berden, Giel; Oomens, Jos; Maitre, Philippe; Steinmetz, Vincent; McNary, Christopher P.; Armentrout, Peter B.; Chow, C. S.; Rodgers, M. T.

2016-06-01

Cisplatin [(NH_3)_2PtCl_2], the first FDA-approved platinum-based anticancer drug, has been widely used in cancer chemotherapy. Its pharmacological mechanism has been identified as its ability to coordinate to genomic DNA with guanine as its major target. Amino acid-linked cisplatin derivatives are being investigated as alternatives for cisplatin that may exhibit altered binding selectivity such as that found for ornithine-linked cisplatin (Ornplatin, [(Orn)PtCl_2]), which exhibits a preference for adenine over guanine in RNA. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and complementary electronic structure calculations are performed on a series of Ornplatin complexes to elucidate the nature of binding of the Orn amino acid to the Pt center and how that binding is influenced by the local environment. The complexes examined in the work include: [(Orn-H)PtCl_2]-, [(Orn)PtCl]+, [(Orn)Pt(H_2O)Cl]+, and [(Orn)PtCl_2+Na]+. In contrast to that found previously for the glycine-linked cisplatin complex (Glyplatin), which binds via the backbone amino and carboxylate groups, binding of Orn in these complexes is found to involve both the backbone and sidechain amino groups. Extensive broadening of the IRMPD spectrum for the [(Orn)Pt(H_2O)Cl]+ complex suggests that either multiple structures are contributing to the measured spectrum or strong intra-molecular hydrogen-binding interactions are present. The results for Ornplatin lead to an interesting discussion about the differences in selectivity and reactivity versus cisplatin.

Structure elucidation of a novel oligosaccharide (Medalose) from camel milk

NASA Astrophysics Data System (ADS)

Gangwar, Lata; Singh, Rinku; Deepak, Desh

2018-02-01

Free oligosaccharides are the third most abundant solid component in milk after lactose and lipids. The study of milk oligosaccharides indicate that nutrients are not only benefits the infant's gut but also perform a number of other functions which include stimulation of growth, receptor analogues to inhibit binding of pathogens and substances that promote postnatal brain development. Surveys reveal that camel milk oligosaccharides possess varied biological activities that help in the treatment of diabetes, asthma, anaemia, piles and also a food supplement to milking mothers. In this research, camel milk was selected for its oligosaccharide contents, which was then processed by Kobata and Ginsburg method followed by the HPLC and CC techniques. Structure elucidation of isolated compound was done by the chemical degradation, chemical transformation and comparison of chemical shift of NMR data of natural and acetylated oligosaccharide structure reporter group theory, the 1H, 13C NMR, 2D-NMR (COSY, TOCSY and HSQC) techniques, and mass spectrometry. The structure was elucidated as under: MEDALOSE

A substrate selectivity and inhibitor design lesson from the PDE10-cAMP crystal structure: a computational study.

PubMed

Lau, Justin Kai-Chi; Li, Xiao-Bo; Cheng, Yuen-Kit

2010-04-22

Phosphodiesterases (PDEs) catalyze the hydrolysis of second messengers cAMP and cGMP in regulating many important cellular signals and have been recognized as important drug targets. Experimentally, a range of specificity/selectivity toward cAMP and cGMP is well-known for the individual PDE families. The study reported here reveals that PDEs might also exhibit selectivity toward conformations of the endogenous substrates cAMP and cGMP. Molecular dynamics simulations and free energy study have been applied to study the binding of the cAMP torsional conformers about the glycosyl bond in PDE10A2. The computational results elucidated that PDE10A2 is energetically more favorable in complex with the syn cAMP conformer (as reported in the crystal structure) and the binding of anti cAMP to PDE10A2 would lead to either a nonreactive configuration or significant perturbation on the catalytic pocket of the enzyme. This experimentally inaccessible information provides important molecular insights for the development of effective PDE10 ligands.

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

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

PubMed

Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

2007-06-01

To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27

PubMed Central

Balana, Bartosz; Maslennikov, Innokentiy; Kwiatkowski, Witek; Stern, Kalyn M.; Bahima, Laia; Choe, Senyon; Slesinger, Paul A.

2011-01-01

G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases. PMID:21422294

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Development of a carbazole-based fluorescence probe for G-quadruplex DNA: The importance of side-group effect on binding specificity

NASA Astrophysics Data System (ADS)

Wang, Ming-Qi; Ren, Gui-Ying; Zhao, Shuang; Lian, Guang-Chang; Chen, Ting-Ting; Ci, Yang; Li, Hong-Yao

2018-06-01

G-quadruplex DNAs are highly prevalent in the human genome and involved in many important biological processes. However, many aspects of their biological mechanism and significance still need to be elucidated. Therefore, the development of fluorescent probes for G-quadruplex detection is important for the basic research. We report here on the development of small molecular dyes designed on the basis of carbazole scaffold by introducing styrene-like substituents at its 9-position, for the purpose of G-quadruplex recognition. Results revealed that the side group on the carbazole scaffold was very important for their ability to selectively recognize G-quadruplex DNA structures. 1a with the pyridine side group displayed excellent fluorescence signal turn-on property for the specific discrimination of G-quadruplex DNAs against other nucleic acids. The characteristics of 1a were further investigated with UV-vis spectrophotometry, fluorescence, circular dichroism, FID assay and molecular docking to validate the selectivity, sensitivity and detailed binding mode toward G-quadruplex DNAs.

The selective autophagy receptor p62 forms a flexible filamentous helical scaffold.

PubMed

Ciuffa, Rodolfo; Lamark, Trond; Tarafder, Abul K; Guesdon, Audrey; Rybina, Sofia; Hagen, Wim J H; Johansen, Terje; Sachse, Carsten

2015-05-05

The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain

PubMed Central

Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J.; Polo, Simona

2016-01-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological role remains unknown. Here we identified an isoform-specific regulatory helix, named α2-linker that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a novel clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, where alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort for tumor cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles. PMID:26950368

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

PubMed

Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

2016-04-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

Elucidation of the binding preferences of peptide recognition modules: SH3 and PDZ domains.

PubMed

Teyra, Joan; Sidhu, Sachdev S; Kim, Philip M

2012-08-14

Peptide-binding domains play a critical role in regulation of cellular processes by mediating protein interactions involved in signalling. In recent years, the development of large-scale technologies has enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. These efforts have provided significant insights into the binding specificities of these modular domains. Many research groups have taken advantage of this unprecedented volume of specificity data and have developed a variety of new algorithms for the prediction of binding specificities of peptide-binding domains and for the prediction of their natural binding targets. This knowledge has also been applied to the design of synthetic peptide-binding domains in order to rewire protein-protein interaction networks. Here, we describe how these experimental technologies have impacted on our understanding of peptide-binding domain specificities and on the elucidation of their natural ligands. We discuss SH3 and PDZ domains as well characterized examples, and we explore the feasibility of expanding high-throughput experiments to other peptide-binding domains. Copyright © 2012. Published by Elsevier B.V.

Structural insights into selective agonist actions of tamoxifen on human estrogen receptor alpha.

PubMed

Chakraborty, Sandipan; Biswas, Pradip Kumar

2014-08-01

Tamoxifen-an anti-estrogenic ligand in breast tissues used as a first-line treatment in estrogen receptor (ER)-positive breast cancers-is associated with the development of resistance followed by resumption of tumor growth in about 30 % of cases. Whether tamoxifen assists in proliferation in such cases or whether any ligand-independent pathway to transcription exists is not fully understood; also, no ERα mutants have been detected so far that could lead to tamoxifen resistance. Using in silico conformational analysis of the ERα ligand binding domain (LBD), in the absence and presence of selective agonist (diethylstilbestrol; DES), antagonist (Faslodex; ICI), and selective estrogen receptor modulator (SERM; 4-hydroxy tamoxifen; 4-OHT) ligands, we have elucidated ligand-responsive structural modulations of the ERα-LBD dimer in its agonist and antagonist complexes to address the issue of "tamoxifen resistance". DES and ICI were found to stabilize the dimer in their agonist and antagonist conformations, respectively. The ERα-LBD dimer without the presence of any bound ligand also led to a stable structure in agonist conformation. However, binding of 4-OHT to the antagonist structure led to a flexible conformation allowing the protein to visit conformations populated by agonists as was evident from principal component analysis and radius of gyration plots. Further, the relaxed conformations of the 4-OHT bound protein exhibited a diminished size of the co-repressor binding pocket in the LBD, thus signaling a partial blockage of the co-repressor binding motif. Thus, the ability of 4-OHT-bound ERα-LBD to assume flexible conformations visited by agonists and reduced co-repressor binding surface at the LBD provide crucial structural insights into tamoxifen-resistance that complement our existing understanding.

Probing the behavior of bovine serum albumin upon binding to atenolol: insights from spectroscopic and molecular docking approaches.

PubMed

Jiang, Tuo-Ying; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi; Shi, Jie-Hua

2018-04-01

Molecular interaction of atenolol, a selective β 1 receptor antagonist with the major carrier protein, bovine serum albumin (BSA), was investigated under imitated physiological conditions (pH 7.4) by means of fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular modeling studies. The steady-state fluorescence spectra manifested that static type, due to formation of the atenolol-BSA complex, was the dominant mechanism for fluorescence quenching. The characteristic information about the binding interaction of atenolol with BSA in terms of binding constant (K b ) were determined by the UV-vis absorption titration, and were found to be in the order of 10 3  M -1 at different temperatures, indicating the existence of a weak binding in this system. Thermodynamic analysis revealed that the binding process was primarily mediated by van der Waals force and hydrogen bonds due to the negative sign for enthalpy change (ΔH 0 ), entropy change (ΔS 0 ). The molecular docking results elucidated that atenolol preferred binding on the site II of BSA according to the findings observed in competitive binding experiments. Moreover, via alterations in synchronous fluorescence, three-dimensional fluorescence and FT-IR spectral properties, it was concluded that atenolol could arouse slight configurational and micro-environmental changes of BSA.

Descriptors and Thermodynamic Limitations of Electrocatalytic Carbon Dioxide Reduction on Rutile Oxide Surfaces.

PubMed

Bhowmik, Arghya; Vegge, Tejs; Hansen, Heine A

2016-11-23

A detailed understanding of the electrochemical reduction of CO 2 into liquid fuels on rutile metal oxide surfaces is developed by using DFT calculations. We consider oxide overlayer structures on RuO 2 (1 1 0) surfaces as model catalysts to elucidate the trends and limitations in the CO 2 reduction reaction (CO2RR) based on thermodynamic analysis. We aim to specify the requirements for CO2RR catalysts to establish adsorbate scaling relations and use these to derive activity volcanoes. Computational results show that the OH* binding free energy is a good descriptor of the thermodynamic limitations and it defines the left leg of the activity volcano for CO2RR. HCOOH* is a key intermediate for products formed through further reduction, for example, methanediol, methanol, and methane. The surfaces that do not bind HCOOH* are selective towards formic acid (HCOOH) production, but hydrogen evolution limits their suitability. We determine the ideal binding free energy for H* and OH* to facilitate selective CO2RR over H 2 /CO evolution to be ΔG B [H]>0.5 eV and -0.5 eV<ΔG B [OH]<0.1 eV. The Re-containing overlayers considered in this work display excellent promise for selectivity, although they are active at a highly reducing potential. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stereo-selectivity of human serum albumin to enantiomeric and isoelectronic pollutants dissected by spectroscopy, calorimetry and bioinformatics.

PubMed

Ahmad, Ejaz; Rabbani, Gulam; Zaidi, Nida; Singh, Saurabh; Rehan, Mohd; Khan, Mohd Moin; Rahman, Shah Kamranur; Quadri, Zainuddin; Shadab, Mohd; Ashraf, Mohd Tashfeen; Subbarao, Naidu; Bhat, Rajiv; Khan, Rizwan Hasan

2011-01-01

1-naphthol (1N), 2-naphthol (2N) and 8-quinolinol (8H) are general water pollutants. 1N and 2N are the configurational enantiomers and 8H is isoelectronic to 1N and 2N. These pollutants when ingested are transported in the blood by proteins like human serum albumin (HSA). Binding of these pollutants to HSA has been explored to elucidate the specific selectivity of molecular recognition by this multiligand binding protein. The association constants (K(b)) of these pollutants to HSA were moderate (10(4)-10(5) M(-1)). The proximity of the ligands to HSA is also revealed by their average binding distance, r, which is estimated to be in the range of 4.39-5.37 nm. The binding free energy (ΔG) in each case remains effectively the same for each site because of enthalpy-entropy compensation (EEC). The difference observed between ΔC(p) (exp) and ΔC(p) (calc) are suggested to be caused by binding-induced flexibility changes in the HSA. Efforts are also made to elaborate the differences observed in binding isotherms obtained through multiple approaches of calorimetry, spectroscopy and bioinformatics. We suggest that difference in dissociation constants of pollutants by calorimetry, spectroscopic and computational approaches could correspond to occurrence of different set of populations of pollutants having different molecular characteristics in ground state and excited state. Furthermore, our observation of enhanced binding of pollutants (2N and 8H) in the presence of hemin signifies that ligands like hemin may enhance the storage period of these pollutants in blood that may even facilitate the ill effects of these pollutants.

Divalent ions are potential permeating blockers of the non-selective NaK ion channel: combined QM and MD based investigations.

PubMed

Sadhu, Biswajit; Sundararajan, Mahesh; Bandyopadhyay, Tusar

2017-10-18

The bacterial NaK ion channel is distinctly different from other known ion channels due to its inherent non-selective feature. One of the unexplored and rather interesting features is its ability to permeate divalent metal ions (such as Ca 2+ and Ba 2+ ) and not monovalent alkali metal ions. Several intriguing questions about the energetics and structural aspects still remain unanswered. For instance, what causes Ca 2+ to permeate as well as block the selectivity filter (SF) of the NaK ion channel and act as a "permeating blocker"? How and at what energetic cost does another chemical congener, Sr 2+ , as well as Ba 2+ , a potent blocker of the K + ion channel, permeate through the SF of the NaK ion channel? Finally, how do their translocation energetics differ from those of monovalent ions such as K + ? Here, in an attempt to address these outstanding issues, we elucidate the structure, binding and selectivity of divalent ions (Ca 2+ , Sr 2+ and Ba 2+ ) as they permeate through the SF of the NaK ion channel using all-atom molecular dynamics simulations and density functional theory based calculations. We unveil mechanistic insight into this translocation event using well-tempered metadynamics simulations in a polarizable environment using the mean-field model of water and incorporating electronic continuum corrections for ions via charge rescaling. The results show that, akin to K + coordination, Sr 2+ and Ba 2+ bind at the SF in a very similar fashion and remain octa-coordinated at all sites. Interestingly, differing from its local hydration structure, Ca 2+ interacts with eight carbonyls to remain at the middle of the S3 site. Furthermore, the binding of divalent metals at SF binding sites is more favorable than the binding of K + . However, their permeation through the extracellular entrance faces a considerably higher energetic barrier compared to that for K + , which eventually manifests their inherent blocking feature.

Molecular Docking, Molecular Dynamics Simulations, Computational Screening to Design Quorum Sensing Inhibitors Targeting LuxP of Vibrio harveyi and Its Biological Evaluation.

PubMed

Rajamanikandan, Sundaraj; Jeyakanthan, Jeyaraman; Srinivasan, Pappu

2017-01-01

Quorum sensing (QS) plays an important role in the biofilm formation, production of virulence factors and stress responses in Vibrio harveyi. Therefore, interrupting QS is a possible approach to modulate bacterial behavior. In the present study, three docking protocols, such as Rigid Receptor Docking (RRD), Induced Fit Docking (IFD), and Quantum Polarized Ligand Docking (QPLD) were used to elucidate the binding mode of boronic acid derivatives into the binding pocket of LuxP protein in V. harveyi. Among the three docking protocols, IFD accurately predicted the correct binding mode of the studied inhibitors. Molecular dynamics (MD) simulations of the protein-ligand complexes indicates that the inter-molecular hydrogen bonds formed between the protein and ligand complex remains stable during the simulation time. Pharmacophore and shape-based virtual screening were performed to find selective and potent compounds from ChemBridge database. Five hit compounds were selected and subjected to IFD and MD simulations to validate the binding mode. In addition, enrichment calculation was performed to discriminate and separate active compounds from the inactive compounds. Based on the computational studies, the potent Bicyclo [2.2.1] hept-5-ene-2,3-dicarboxylic acid-2,6-dimethylpyridine 1-oxide (ChemBridge_5144368) was selected for in vitro assays. The compound exhibited dose dependent inhibition in bioluminescence and also inhibits biofilm formation in V. harveyi to the level of 64.25 %. The result from the study suggests that ChemBridge_5144368 could serve as an anti-quorum sensing molecule for V. harveyi.

Comprehensive comparative analysis and identification of RNA-binding protein domains: multi-class classification and feature selection.

PubMed

Jahandideh, Samad; Srinivasasainagendra, Vinodh; Zhi, Degui

2012-11-07

RNA-protein interaction plays an important role in various cellular processes, such as protein synthesis, gene regulation, post-transcriptional gene regulation, alternative splicing, and infections by RNA viruses. In this study, using Gene Ontology Annotated (GOA) and Structural Classification of Proteins (SCOP) databases an automatic procedure was designed to capture structurally solved RNA-binding protein domains in different subclasses. Subsequently, we applied tuned multi-class SVM (TMCSVM), Random Forest (RF), and multi-class ℓ1/ℓq-regularized logistic regression (MCRLR) for analysis and classifying RNA-binding protein domains based on a comprehensive set of sequence and structural features. In this study, we compared prediction accuracy of three different state-of-the-art predictor methods. From our results, TMCSVM outperforms the other methods and suggests the potential of TMCSVM as a useful tool for facilitating the multi-class prediction of RNA-binding protein domains. On the other hand, MCRLR by elucidating importance of features for their contribution in predictive accuracy of RNA-binding protein domains subclasses, helps us to provide some biological insights into the roles of sequences and structures in protein-RNA interactions.

Computational Optimization and Characterization of Molecularly Imprinted Polymers

NASA Astrophysics Data System (ADS)

Terracina, Jacob J.

Molecularly imprinted polymers (MIPs) are a class of materials containing sites capable of selectively binding to the imprinted target molecule. Computational chemistry techniques were used to study the effect of different fabrication parameters (the monomer-to-target ratios, pre-polymerization solvent, temperature, and pH) on the formation of the MIP binding sites. Imprinted binding sites were built in silico for the purposes of better characterizing the receptor - ligand interactions. Chiefly, the sites were characterized with respect to their selectivities and the heterogeneity between sites. First, a series of two-step molecular mechanics (MM) and quantum mechanics (QM) computational optimizations of monomer -- target systems was used to determine optimal monomer-to-target ratios for the MIPs. Imidazole- and xanthine-derived target molecules were studied. The investigation included both small-scale models (one-target) and larger scale models (five-targets). The optimal ratios differed between the small and larger scales. For the larger models containing multiple targets, binding-site surface area analysis was used to evaluate the heterogeneity of the sites. The more fully surrounded sites had greater binding energies. Molecular docking was then used to measure the selectivities of the QM-optimized binding sites by comparing the binding energies of the imprinted target to that of a structural analogue. Selectivity was also shown to improve as binding sites become more fully encased by the monomers. For internal sites, docking consistently showed selectivity favoring the molecules that had been imprinted via QM geometry optimizations. The computationally imprinted sites were shown to exhibit size-, shape-, and polarity-based selectivity. This represented a novel approach to investigate the selectivity and heterogeneity of imprinted polymer binding sites, by applying the rapid orientation screening of MM docking to the highly accurate QM-optimized geometries. Next, we sought to computationally construct and investigate binding sites for their enantioselectivity. Again, a two-step MM [special characters removed] QM optimization scheme was used to "computationally imprint" chiral molecules. Using docking techniques, the imprinted binding sites were shown to exhibit an enantioselective preference for the imprinted molecule over its enantiomer. Docking of structurally similar chiral molecules showed that the sites computationally imprinted with R- or S-tBOC-tyrosine were able to differentiate between R- and S-forms of other tyrosine derivatives. The cross-enantioselectivity did not hold for chiral molecules that did not share the tyrosine H-bonding functional group orientations. Further analysis of the individual monomer - target interactions within the binding site led us to conclude that H-bonding functional groups that are located immediately next to the target's chiral center, and therefore spatially fixed relative to the chiral center, will have a stronger contribution to the enantioselectivity of the site than those groups separated from the chiral center by two or more rotatable bonds. These models were the first computationally imprinted binding sites to exhibit this enantioselective preference for the imprinted target molecules. Finally, molecular dynamics (MD) was used to quantify H-bonding interactions between target molecules, monomers, and solvents representative of the pre-polymerization matrix. It was found that both target dimerization and solvent interference decrease the number of monomer - target H-bonds present. Systems were optimized via simulated annealing to create binding sites that were then subjected to molecular docking analysis. Docking showed that the presence of solvent had a detrimental effect on the sensitivity and selectivity of the sites, and that solvents with more H-bonding capabilities were more disruptive to the binding properties of the site. Dynamic simulations also showed that increasing the temperature of the solution can significantly decrease the number of H-bonds formed between the targets and monomers. It is believed that the monomer - target complexes formed within the pre-polymerization matrix are translated into the selective binding cavities formed during polymerization. Elucidating the nature of these interactions in silico improves our understanding of MIPs, ultimately allowing for more optimized sensing materials.

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.

PubMed

Wise, Jillian F; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K; Tao, Rong-Hua; Sabichi, Anita L; Ao, Xue; Maeng, Hoyoung; Samaniego, Felipe

2013-06-06

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex

PubMed Central

Wise, Jillian F.; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K.; Tao, Rong-Hua; Sabichi, Anita L.; Ao, Xue; Maeng, Hoyoung

2013-01-01

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL–Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target. PMID:23599269

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

PubMed Central

Kalra, Priya; Dhiman, Abhijeet; Cho, William C.; Bruno, John G.; Sharma, Tarun K.

2018-01-01

Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays. PMID:29868605

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity.

PubMed

Kalra, Priya; Dhiman, Abhijeet; Cho, William C; Bruno, John G; Sharma, Tarun K

2018-01-01

Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays.

Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.

PubMed

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E; Przytycka, Teresa M

2012-06-15

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.

Adaptor proteins in protein kinase C-mediated signal transduction.

PubMed

Schechtman, D; Mochly-Rosen, D

2001-10-01

Spatial and temporal organization of signal transduction is essential in determining the speed and precision by which signaling events occur. Adaptor proteins are key to organizing signaling enzymes near their select substrates and away from others in order to optimize precision and speed of response. Here, we describe the role of adaptor proteins in determining the specific function of individual protein kinase C (PKC) isozymes. These isozyme-selective proteins were called collectively RACKs (receptors for activated C-kinase). The role of RACKs in PKC-mediated signaling was determined using isozyme-specific inhibitors and activators of the binding of each isozyme to its respective RACK. In addition to anchoring activated PKC isozymes, RACKs anchor other signaling enzymes. RACK1, the anchoring protein for activated betaIIPKC, binds for example, Src tyrosine kinase, integrin, and phosphodiesterase. RACK2, the epsilonPKC-specific RACK, is a coated-vesicle protein and thus is involved in vesicular release and cell-cell communication. Therefore, RACKs are not only adaptors for PKC, but also serve as adaptor proteins for several other signaling enzymes. Because at least some of the proteins that bind to RACKs, including PKC itself, regulate cell growth, modulating their interactions with RACKs may help elucidate signaling pathways leading to carcinogenesis and could result in the identification of novel therapeutic targets.

Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site.

PubMed

Paiz-Candia, Bertin; Islas, Angel A; Sánchez-Solano, Alfredo; Mancilla-Simbro, Claudia; Scior, Thomas; Millan-PerezPeña, Lourdes; Salinas-Stefanon, Eduardo M

2017-02-05

Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na + channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na + channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Na v 1.4 currents (IC 50 =60μM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the β1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Na v 1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na + channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a carbazole-based fluorescence probe for G-quadruplex DNA: The importance of side-group effect on binding specificity.

PubMed

Wang, Ming-Qi; Ren, Gui-Ying; Zhao, Shuang; Lian, Guang-Chang; Chen, Ting-Ting; Ci, Yang; Li, Hong-Yao

2018-06-15

G-quadruplex DNAs are highly prevalent in the human genome and involved in many important biological processes. However, many aspects of their biological mechanism and significance still need to be elucidated. Therefore, the development of fluorescent probes for G-quadruplex detection is important for the basic research. We report here on the development of small molecular dyes designed on the basis of carbazole scaffold by introducing styrene-like substituents at its 9-position, for the purpose of G-quadruplex recognition. Results revealed that the side group on the carbazole scaffold was very important for their ability to selectively recognize G-quadruplex DNA structures. 1a with the pyridine side group displayed excellent fluorescence signal turn-on property for the specific discrimination of G-quadruplex DNAs against other nucleic acids. The characteristics of 1a were further investigated with UV-vis spectrophotometry, fluorescence, circular dichroism, FID assay and molecular docking to validate the selectivity, sensitivity and detailed binding mode toward G-quadruplex DNAs. Copyright © 2018 Elsevier B.V. All rights reserved.

Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition

PubMed Central

Wu, Wen-I; Voegtli, Walter C.; Sturgis, Hillary L.; Dizon, Faith P.; Vigers, Guy P. A.; Brandhuber, Barbara J.

2010-01-01

AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones. PMID:20886116

Molecular simulations of multimodal ligand-protein binding: elucidation of binding sites and correlation with experiments.

PubMed

Freed, Alexander S; Garde, Shekhar; Cramer, Steven M

2011-11-17

Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

Interaction of small molecules with double-stranded RNA: spectroscopic, viscometric, and calorimetric study of hoechst and proflavine binding to PolyCG structures.

PubMed

Sinha, Rangana; Hossain, Maidul; Kumar, Gopinatha Suresh

2009-04-01

Design and synthesis of new small molecules binding to double-stranded RNA necessitate complete understanding of the molecular aspects of the binding of many existing molecules. Toward this goal, in this work we evaluated the biophysical aspects of the interaction of a DNA intercalator (proflavine) and a minor groove binder (hoechst 33258) with two polymorphic forms of polyCG, namely, the right-handed Watson-Crick base paired A-form and the left-handed Hoogsteen base paired H(L)-form, by absorption, fluorescence, and viscometry experiments. The energetics of the interaction of these molecules with the RNA structures has also been elucidated by isothermal titration calorimetry (ITC). Results suggest that proflavine strongly intercalates in both forms of polyCG, whereas hoechst shows mainly groove-binding modes. The binding of both drugs to both forms of RNA resulted in significant conformational change to the RNA structure with the bound molecules being placed in the chiral RNA helix. ITC profiles for both proflavine and hoechst show two binding sites. Binding of proflavine to both forms of RNA is endothermic and entropy driven in the first site and exothermic and enthalpy driven in the second site, whereas hoechst binding to both forms of RNA is exothermic and enthalpy driven in the first site and endothermic and entropy driven in the second site. This study suggests that the binding affinity characteristics and energetics of interaction of these DNA binding molecules with the RNA conformations are significantly different and may serve as data for future development of effective structure-selective RNA-based drugs.

Elucidation of the factors affecting the oxidative activity of Acremonium sp. HI-25 ascorbate oxidase by an electrochemical approach

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

Murata, Kenichi; Nakamura, Nobuhumi; Ohno, Hiroyuki

Steady-state kinetics of Acremonium sp. HI-25 ascorbate oxidase toward p-hydroquinone derivatives have been examined by using an electrochemical analysis based on the theory of steady-state bioelectrocatalysis. The electrochemical technique has enabled one to examine the influence of electronic and chemical properties of substrates on the activity. It was proven that the oxidative activity of ascorbate oxidase was dominated by the highly selective substrate-binding affinity based on electrostatic interaction beyond the one-electron redox potential difference between ascorbate oxidase's type 1 copper site and substrate.

A 90-Kilodalton Endothelial Cell Molecule Mediating Lymphocyte Binding in Humans

NASA Astrophysics Data System (ADS)

Salmi, Marko; Jalkanen, Sirpa

1992-09-01

Interactions between leukocyte surface receptors and their ligands on vascular endothelial cells control lymphocyte traffic between the blood and various lymphoid organs, as well as extravasation of leukocytes into sites of inflammation. A heretofore undescribed 90-kilodalton human endothelial cell adhesion molecule (VAP-1) defined by a monoclonal antibody 1B2 is described. The expression pattern, molecular mass, functional properties, and an amino-terminal amino acid sequence define VAP-1 as an endothelial ligand for lymphocytes. VAP-1 helps to elucidate the complex heterotypic cell interactions that direct tissue-selective lymphocyte migration in man.

Insights into the structural/conformational requirements of cytotoxic oxadiazoles as potential chemotherapeutic target binding agents

NASA Astrophysics Data System (ADS)

Alikhani, Radin; Razzaghi-Asl, Nima; Ramazani, Ali; Hosseinzadeh, Zahra

2018-07-01

A few novel previously synthesized 2,5-disubstituted 1,3,4-oxadiazoles with cytotoxic activity (1-17) were subjected to combined docking/quantum mechanical studies against chemotherapeutic targets. Selected macromolecular targets were those that were previously known to be inhibited by 1,3,4-oxadiazoles. Within this work, favorable binding modes/affinities of the oxadiazoles toward validated cancer targets were elucidated. Some oxadiazole structures exhibited ΔGbs comparable to or stronger than crystallographic ligands that were previously demonstrated to inhibit such targets. On the basis of obtained results, a general structure activity/binding relationship (SAR/SBR) was developed and a few 2,5-disubstituted 1,3,4-oxadiazole structures were proposed and virtually validated as potential cytotoxic candidates. To get more insight into structure binding relationship of candidate molecules within best correlated targets, docked conformation of the best in silico in vitro correlated oxadiazole structure was analyzed in terms of intermolecular binding energy components by functional B3LYP in association with split valence basis set using polarization functions (Def2-SVP). We believe that such modeling studies may be complementary to our previous results on the synthesis and cytotoxicity assessment of novel 1,3,4-oxadiazole derivatives through extending the scope of privileged structures toward designing new potential anti-tumor compounds.

PDZ Ligand Binding-Induced Conformational Coupling of the PDZ-SH3-GK Tandems in PSD-95 Family MAGUKs.

PubMed

Zeng, Menglong; Ye, Fei; Xu, Jia; Zhang, Mingjie

2018-01-05

Discs large (DLG) MAGUKs are abundantly expressed in glutamatergic synapses, crucial for synaptic transmission, and plasticity by anchoring various postsynaptic components including glutamate receptors, downstream scaffold proteins and signaling enzymes. Different DLG members have shared structures and functions, but also contain unique features. How DLG family proteins function individually and cooperatively is largely unknown. Here, we report that PSD-95 PDZ3 directly couples with SH3-GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization. Aided by sortase-mediated protein ligation and selectively labeling, we elucidated the PDZ3/SH3-GK conformational coupling mechanism using NMR spectroscopy. We further demonstrated that PSD-93, but not SAP102, can also undergo PDZ3 ligand binding-induced conformational coupling with SH3-GK and form homo-oligomers. Interestingly, PSD-95 and PSD-93 can also form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-N-methyl-d-aspartate receptor synaptic signaling complex. Finally, we provide evidence showing that ligand binding-induced conformational coupling between PDZ and SH3-GK is a common feature for other MAGUKs including CASK and PALS1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Elucidation of the binding mechanism of renin using a wide array of computational techniques and biological assays.

PubMed

Tzoupis, Haralambos; Leonis, Georgios; Avramopoulos, Aggelos; Reis, Heribert; Czyżnikowska, Żaneta; Zerva, Sofia; Vergadou, Niki; Peristeras, Loukas D; Papavasileiou, Konstantinos D; Alexis, Michael N; Mavromoustakos, Thomas; Papadopoulos, Manthos G

2015-11-01

We investigate the binding mechanism in renin complexes, involving three drugs (remikiren, zankiren and enalkiren) and one lead compound, which was selected after screening the ZINC database. For this purpose, we used ab initio methods (the effective fragment potential, the variational perturbation theory, the energy decomposition analysis, the atoms-in-molecules), docking, molecular dynamics, and the MM-PBSA method. A biological assay for the lead compound has been performed to validate the theoretical findings. Importantly, binding free energy calculations for the three drug complexes are within 3 kcal/mol of the experimental values, thus further justifying our computational protocol, which has been validated through previous studies on 11 drug-protein systems. The main elements of the discovered mechanism are: (i) minor changes are induced to renin upon drug binding, (ii) the three drugs form an extensive network of hydrogen bonds with renin, whilst the lead compound presented diminished interactions, (iii) ligand binding in all complexes is driven by favorable van der Waals interactions and the nonpolar contribution to solvation, while the lead compound is associated with diminished van der Waals interactions compared to the drug-bound forms of renin, and (iv) the environment (H2O/Na(+)) has a small effect on the renin-remikiren interaction. Copyright © 2015 Elsevier Inc. All rights reserved.

AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA

PubMed Central

Götz, Frank; Roske, Yvette; Schulz, Maike Svenja; Autenrieth, Karolin; Bertinetti, Daniela; Faelber, Katja; Zühlke, Kerstin; Kreuchwig, Annika; Kennedy, Eileen J.; Krause, Gerd; Daumke, Oliver; Herberg, Friedrich W.; Heinemann, Udo; Klussmann, Enno

2016-01-01

A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18β bound to the D/D domain of the regulatory RIIα subunits of PKA. We identified three hydrophilic anchor points in AKAP18β outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions. PMID:27102985

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4.

PubMed

Sehar, Ujala; Mehmood, Muhammad Aamer; Hussain, Khadim; Nawaz, Salman; Nadeem, Shahid; Siddique, Muhammad Hussnain; Nadeem, Habibullah; Gull, Munazza; Ahmad, Niaz; Sohail, Iqra; Gill, Saba Shahid; Majeed, Summera

2013-01-01

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

NASA Astrophysics Data System (ADS)

Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

2014-12-01

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

The crystal structure of the AgamOBP1•Icaridin complex reveals alternative binding modes and stereo-selective repellent recognition.

PubMed

Drakou, Christina E; Tsitsanou, Katerina E; Potamitis, Constantinos; Fessas, Dimitrios; Zervou, Maria; Zographos, Spyros E

2017-01-01

Anopheles gambiae Odorant Binding Protein 1 in complex with the most widely used insect repellent DEET, was the first reported crystal structure of an olfactory macromolecule with a repellent, and paved the way for OBP1-structure-based approaches for discovery of new host-seeking disruptors. In this work, we performed STD-NMR experiments to directly monitor and verify the formation of a complex between AgamOBP1 and Icaridin, an efficient DEET alternative. Furthermore, Isothermal Titration Calorimetry experiments provided evidence for two Icaridin-binding sites with different affinities (Kd = 0.034 and 0.714 mM) and thermodynamic profiles of ligand binding. To elucidate the binding mode of Icaridin, the crystal structure of AgamOBP1•Icaridin complex was determined at 1.75 Å resolution. We found that Icaridin binds to the DEET-binding site in two distinct orientations and also to a novel binding site located at the C-terminal region. Importantly, only the most active 1R,2S-isomer of Icaridin's equimolar diastereoisomeric mixture binds to the AgamOBP1 crystal, providing structural evidence for the possible contribution of OBP1 to the stereoselectivity of Icaridin perception in mosquitoes. Structural analysis revealed two ensembles of conformations differing mainly in spatial arrangement of their sec-butyl moieties. Moreover, structural comparison with DEET indicates a common recognition mechanism for these structurally related repellents. Ligand interactions with both sites and binding modes were further confirmed by 2D 1 H- 15 N HSQC NMR spectroscopy. The identification of a novel repellent-binding site in AgamOBP1 and the observed structural conservation and stereoselectivity of its DEET/Icaridin-binding sites open new perspectives for the OBP1-structure-based discovery of next-generation insect repellents.

Energetic factors determining the binding of type I inhibitors to c-Met kinase: experimental studies and quantum mechanical calculations.

PubMed

Yu, Zhe; Ma, Yu-chi; Ai, Jing; Chen, Dan-qi; Zhao, Dong-mei; Wang, Xin; Chen, Yue-lei; Geng, Mei-yu; Xiong, Bing; Cheng, Mao-sheng; Shen, Jing-Kang

2013-11-01

To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory (SAPT) was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich π-π interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

NASA Astrophysics Data System (ADS)

Dong, Jinlan; Bruening, Merlin L.

2015-07-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

PubMed

Dong, Jinlan; Bruening, Merlin L

2015-01-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Structural characterization of tetranortriterpenes from Pseudrocedrela kotschyi and Trichilia emetica and study of their activity towards the chaperone Hsp90.

PubMed

Piaz, Fabrizio Dal; Malafronte, Nicola; Romano, Adriana; Gallotta, Dario; Belisario, Maria Antonietta; Bifulco, Giuseppe; Gualtieri, Maria Josefine; Sanogo, Rokia; Tommasi, Nunziatina De; Pisano, Claudio

2012-03-01

Investigation of roots extracts Pseudrocedrela kotschyi and Trichilia emetica led to identification of 5 limonoid derivatives, Kotschyins D-H, and 11 known compounds. Their structures were elucidated by extensive 1D and 2D NMR experiments in conjunction with mass spectrometry. A surface plasmon resonance (SPR) approach was adopted to screen their Hsp90 binding capability and kotschyin D showed a significant affinity for the chaperone. Therefore, the characterization of the biological activity of kotschyin D by means of a panel of chemical and biological approaches, including limited proteolysis, molecular docking and biochemical and cellular assays, was performed. Our result indicated this compound as a type of client selective Hsp90 inhibitor, directly binding to the middle domain of the protein and possibly preventing its interaction with the activator of Hsp90 ATPase 1 (Aha1). Copyright © 2011 Elsevier Ltd. All rights reserved.

Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

PubMed

Gruber, David F; Gaffney, Jean P; Mehr, Shaadi; DeSalle, Rob; Sparks, John S; Platisa, Jelena; Pieribone, Vincent A

2015-01-01

We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins.

PubMed

Toleman, Clifford A; Schumacher, Maria A; Yu, Seok-Ho; Zeng, Wenjie; Cox, Nathan J; Smith, Timothy J; Soderblom, Erik J; Wands, Amberlyn M; Kohler, Jennifer J; Boyce, Michael

2018-06-05

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3β/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.

Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

DOE PAGES

Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius; ...

2015-05-07

We report that angiotensin II type 1 receptor (AT 1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT 1R blockers (ARBs), the structural basis for AT 1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT 1R in complex with its selective antagonist ZD7155 atmore » 2.9 Å resolution. The AT 1R-ZD7155 complex structure revealed key structural features ofAT 1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT 1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT 1R structure-function relationship and structure-based drug design.« less

Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

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

Zhang, Haitao; Unal, Hamiyet; Gati, Cornelius

We report that angiotensin II type 1 receptor (AT 1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT 1R blockers (ARBs), the structural basis for AT 1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT 1R in complex with its selective antagonist ZD7155 atmore » 2.9 Å resolution. The AT 1R-ZD7155 complex structure revealed key structural features ofAT 1R and critical interactions for ZD7155 binding. Finally, docking simulations of the clinically used ARBs into the AT 1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT 1R structure-function relationship and structure-based drug design.« less

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver

PubMed Central

2018-01-01

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. PMID:29757144

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver.

PubMed

Matthews, Bryan J; Waxman, David J

2018-05-14

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. © 2018, Matthews et al.

Reverse spin-crossover and high-pressure kinetics of the heme iron center relevant for the operation of heme proteins under deep-sea conditions.

PubMed

Troeppner, Oliver; Lippert, Rainer; Shubina, Tatyana E; Zahl, Achim; Jux, Norbert; Ivanović-Burmazović, Ivana

2014-10-20

By design of a heme model complex with a binding pocket of appropriate size and flexibility, and by elucidating its kinetics and thermodynamics under elevated pressures, some of the pressure effects are demonstrated relevant for operation of heme-proteins under deep-sea conditions. Opposite from classical paradigms of the spin-crossover and reaction kinetics, a pressure increase can cause deceleration of the small-molecule binding to the vacant coordination site of the heme-center in a confined space and stabilize a high-spin state of its Fe center. This reverse high-pressure behavior can be achieved only if the volume changes related to the conformational transformation of the cavity can offset the volume changes caused by the substrate binding. It is speculated that based on these criteria nature could make a selection of structures of heme pockets that assist in reducing metabolic activity and enzymatic side reactions under extreme pressure conditions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of sequence–structure RNA binding motifs for SELEX-derived aptamers

PubMed Central

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E.; Przytycka, Teresa M.

2012-01-01

Motivation: Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. Results: To close this gap we developed, Aptamotif, a computational method for the identification of sequence–structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process. Contact: przytyck@ncbi.nlm.nih.gov, Zuben.Sauna@fda.hhs.gov PMID:22689764

[The significance of Ulex europaeus agglutinin I lectin binding fibers in various muscular diseases].

PubMed

Yatabe, K; Hiraguri, M; Sueishi, M; Takeuchi, M; Nonaka, I; Kawai, M

1998-05-01

In the present study, we have reported that Ulex europaeus agglutinin I (UEA I) lectin labeled muscle fibers in distal myopathy with rimmed vacuole formation (DMRV). UEA I binding to muscle fibers was also observed in a small number of biopsies with inflammatory myopathy, but not in other diseases, including neurogenic muscular atrophies and muscular dystrophies. In order to elucidate the relationship between this UEA I binding, rimmed vacuole formation and active autophagocytosis, we examined the UEA I binding fibers in other myopathies which frequently showed rimmed vacuoles, including adult onset acid maltase deficiency, oculo-pharyngo-distal type myopathy and oculopharyngeal muscular dystrophy. No UEA I lectin labeling fiber was observed in the diseases examined. We then studied UEA I binding behavior on 70 biopsies of inflammatory myopathy to characterize the clinical features of UEA I binding positive patients. UEA I binding fibers were observed in 3 of 28 patients (11%) with other collagen diseases, 11 of 36 (31%) without these disorders, and 2 of 6 (33%) with inclusion body myositis. There were no common clinical histories, complications or laboratory findings among the UEA I binding positive patients. In conclusion, a common process may exist between the muscle fiber degeneration in DMRV and subgroups of inflammatory myopathy patients, but the basic mechanism remains to be elucidated.

The neuroanatomical distribution of oxytocin receptor binding and mRNA in the male rhesus macaque (Macaca mulatta).

PubMed

Freeman, Sara M; Inoue, Kiyoshi; Smith, Aaron L; Goodman, Mark M; Young, Larry J

2014-07-01

The rhesus macaque (Macaca mulatta) is an important primate model for social cognition, and recent studies have begun to explore the impact of oxytocin on social cognition and behavior. Macaques have great potential for elucidating the neural mechanisms by which oxytocin modulates social cognition, which has implications for oxytocin-based pharmacotherapies for psychiatric disorders such as autism and schizophrenia. Previous attempts to localize oxytocin receptors (OXTR) in the rhesus macaque brain have failed due to reduced selectivity of radioligands, which in primates bind to both OXTR and the structurally similar vasopressin 1a receptor (AVPR1A). We have developed a pharmacologically-informed competitive binding autoradiography protocol that selectively reveals OXTR and AVPR1A binding sites in primate brain sections. Using this protocol, we describe the neuroanatomical distribution of OXTR in the macaque. Finally, we use in situ hybridization to localize OXTR mRNA. Our results demonstrate that OXTR expression in the macaque brain is much more restricted than AVPR1A. OXTR is largely limited to the nucleus basalis of Meynert, pedunculopontine tegmental nucleus, the superficial gray layer of the superior colliculus, the trapezoid body, and the ventromedial hypothalamus. These regions are involved in a variety of functions relevant to social cognition, including modulating visual attention, processing auditory and multimodal sensory stimuli, and controlling orienting responses to visual stimuli. These results provide insights into the neural mechanisms by which oxytocin modulates social cognition and behavior in this species, which, like humans, uses vision and audition as the primary modalities for social communication. Copyright © 2014 Elsevier Ltd. All rights reserved.

The neuroanatomical distribution of oxytocin receptor binding and mRNA in the male rhesus macaque (Macaca mulatta)

PubMed Central

Freeman, Sara M.; Inoue, Kiyoshi; Smith, Aaron L.; Goodman, Mark M.; Young, Larry J.

2014-01-01

The rhesus macaque (Macaca mulatta) is an important primate model for social cognition, and recent studies have begun to explore the impact of oxytocin on social cognition and behavior. Macaques have great potential for elucidating the neural mechanisms by which oxytocin modulates social cognition, which has implications for oxytocin-based pharmacotherapies for psychiatric disorders such as autism and schizophrenia. Previous attempts to localize oxytocin receptors (OXTR) in the rhesus macaque brain have failed due to reduced selectivity of radioligands, which in primates bind to both OXTR and the structurally similar vasopressin 1a receptor (AVPR1A). We have developed a pharmacologically-informed competitive binding autoradiography protocol that selectively reveals OXTR and AVPR1A binding sites in primate brain sections. Using this protocol, we describe the neuroanatomical distribution of OXTR in the macaque. Finally, we use in situ hybridization to localize OXTR mRNA. Our results demonstrate that OXTR expression in the macaque brain is much more restricted than AVPR1A. OXTR is largely limited to the nucleus basalis of Meynert, pedunculopontine tegmental nucleus, the superficial gray layer of the superior colliculus, the trapezoid body, and the ventromedial hypothalamus. These regions are involved in a variety of functions relevant to social cognition, including modulating visual attention, processing auditory and multimodal sensory stimuli, and controlling orienting responses to visual stimuli. These results provide insights into the neural mechanisms by which oxytocin modulates social cognition and behavior in this species, which, like humans, uses vision and audition as the primary modalities for social communication. PMID:24845184

Genome-Wide Binding Analysis of the Transcription Activator IDEAL PLANT ARCHITECTURE1 Reveals a Complex Network Regulating Rice Plant Architecture[W

PubMed Central

Lu, Zefu; Yu, Hong; Xiong, Guosheng; Wang, Jing; Jiao, Yongqing; Liu, Guifu; Jing, Yanhui; Meng, Xiangbing; Hu, Xingming; Qian, Qian; Fu, Xiangdong; Wang, Yonghong; Li, Jiayang

2013-01-01

IDEAL PLANT ARCHITECTURE1 (IPA1) is critical in regulating rice (Oryza sativa) plant architecture and substantially enhances grain yield. To elucidate its molecular basis, we first confirmed IPA1 as a functional transcription activator and then identified 1067 and 2185 genes associated with IPA1 binding sites in shoot apices and young panicles, respectively, through chromatin immunoprecipitation sequencing assays. The SQUAMOSA PROMOTER BINDING PROTEIN-box direct binding core motif GTAC was highly enriched in IPA1 binding peaks; interestingly, a previously uncharacterized indirect binding motif TGGGCC/T was found to be significantly enriched through the interaction of IPA1 with proliferating cell nuclear antigen PROMOTER BINDING FACTOR1 or PROMOTER BINDING FACTOR2. Genome-wide expression profiling by RNA sequencing revealed IPA1 roles in diverse pathways. Moreover, our results demonstrated that IPA1 could directly bind to the promoter of rice TEOSINTE BRANCHED1, a negative regulator of tiller bud outgrowth, to suppress rice tillering, and directly and positively regulate DENSE AND ERECT PANICLE1, an important gene regulating panicle architecture, to influence plant height and panicle length. The elucidation of target genes of IPA1 genome-wide will contribute to understanding the molecular mechanisms underlying plant architecture and to facilitating the breeding of elite varieties with ideal plant architecture. PMID:24170127

Elucidation of the Binding Mode of the Carboxyterminal Region of Peptide YY to the Human Y2 Receptor.

PubMed

Xu, Bo; Vasile, Silvana; Østergaard, Søren; Paulsson, Johan F; Pruner, Jasna; Åqvist, Johan; Wulff, Birgitte S; Gutiérrez-de-Terán, Hugo; Larhammar, Dan

2018-04-01

Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y 2 receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment ( 32 TRQRY 36 -amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y 2 receptor. The amidated C-terminus would be stabilized by polar interactions with Gln288 6.55 and Tyr219 5.39 , while Gln130 3.32 contributes to interactions with Q 34 in the peptide and T 32 is close to the tip of TM7 in the receptor. This leaves the core, α -helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y 2 system and can be used as a basis for optimization of Y 2 receptor agonists. Copyright © 2018 by The Author(s).

Antascomicins A, B, C, D and E. Novel FKBP12 binding compounds from a Micromonospora strain.

PubMed

Fehr, T; Sanglier, J J; Schuler, W; Gschwind, L; Ponelle, M; Schilling, W; Wioland, C

1996-03-01

5 novel ascomycin-like compounds, antascomicins A, B, C, D and E were isolated from a strain of Micromonospora. The antascomicins bind strongly to the FK506-binding protein FKBP12 and antagonize the immunosuppressive activity of FK506 and rapamycin. The strain description, fermentation, structure elucidation and biological activity of these compounds are described.

Energetic factors determining the binding of type I inhibitors to c-Met kinase: experimental studies and quantum mechanical calculations

PubMed Central

Yu, Zhe; Ma, Yu-chi; Ai, Jing; Chen, Dan-qi; Zhao, Dong-mei; Wang, Xin; Chen, Yue-lei; Geng, Mei-yu; Xiong, Bing; Cheng, Mao-sheng; Shen, Jing-kang

2013-01-01

Aim: To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. Methods: Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory (SAPT) was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. Results: Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich π–π interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. Conclusion: The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met. PMID:24056705

Group 10 allergens (tropomyosins) from house-dust mites may cause covariation of sensitization to allergens from other invertebrates

PubMed Central

Inam, Muhammad; Ismail, Muhammad; Chaudhary, Farhana Riaz

2012-01-01

Group 10 allergens (tropomyosins) have been assumed to be a major cause of cross-reactivity between house-dust mites (HDMs) and other invertebrates. Despite all of the published data regarding the epidemiology, percent IgE binding and level of sensitization in the population, the role of tropomyosin as a cross-reactive allergen in patients with multiple allergy syndrome still remains to be elucidated. Homology between amino acid sequences reported in allergen databases of selected invertebrate tropomyosins was determined with Der f 10 as the reference allergen. The 66.9 and 54.4% identities were found with selected crustacean and insect species, respectively, whereas only 20.4% identity was seen with mollusks. A similar analysis was performed using reported B-cell IgE-binding epitopes from Met e1 (shrimp allergen) and Bla g7 (cockroach allergen) with other invertebrate tropomyosins. The percent identity in linear sequences was higher than 35% in mites, crustaceans, and cockroaches. The polar and hydrophobic regions in these groups were highly conserved. These findings suggest that tropomyosin may be a major cause of covariation of sensitization between HDMs, crustaceans, and some species of insects and mollusks. PMID:23342293

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody.

PubMed

Boyles, Jeffrey S; Atwell, Shane; Druzina, Zhanna; Heuer, Josef G; Witcher, Derrick R

2016-11-01

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala 41 , Glu 44 , and His 45 . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859. © 2016 The Protein Society.

Interaction of lafutidine in binding to human serum albumin in gastric ulcer therapy: STD-NMR, WaterLOGSY-NMR, NMR relaxation times, Tr-NOESY, molecule docking, and spectroscopic studies.

PubMed

Yang, Hongqin; Huang, Yanmei; He, Jiawei; Li, Shanshan; Tang, Bin; Li, Hui

2016-09-15

In this study, lafutidine (LAF) was used as a model compound to investigate the binding mechanism between antiulcer drugs and human serum albumin (HSA) through various techniques, including STD-NMR, WaterLOGSY-NMR, (1)H NMR relaxation times, tr-NOESY, molecule docking calculation, FT-IR spectroscopy, and CD spectroscopy. The analyses of STD-NMR, which derived relative STD (%) intensities, and WaterLOGSY-NMR, determined that LAF bound to HSA. In particular, the pyridyl group of LAF was in close contact with HSA binding pocket, whereas furyl group had a secondary binding. Competitive STD-NMR and WaterLOGSY-NMR experiments, with warifarin and ibuprofen as site-selective probes, indicated that LAF preferentially bound to site II in the hydrophobic subdomains IIIA of HSA. The bound conformation of LAF at the HSA binding site was further elucidated by transferred NOE effect (tr-NOESY) experiment. Relaxation experiments provided quantitative information about the relationship between the affinity and structure of LAF. The molecule docking simulations conducted with AutoDock and the restraints derived from STD results led to three-dimensional models that were consistent with the NMR spectroscopic data. The presence of hydrophobic forces and hydrogen interactions was also determined. Additionally, FT-IR and CD spectroscopies showed that LAF induced secondary structure changes of HSA. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural basis of gene regulation by the Grainyhead/CP2 transcription factor family

PubMed Central

Ming, Qianqian; Roske, Yvette; Schuetz, Anja; Walentin, Katharina; Ibraimi, Ibraim; Schmidt-Ott, Kai M

2018-01-01

Abstract Grainyhead (Grh)/CP2 transcription factors are highly conserved in multicellular organisms as key regulators of epithelial differentiation, organ development and skin barrier formation. In addition, they have been implicated as being tumor suppressors in a variety of human cancers. Despite their physiological importance, little is known about their structure and DNA binding mode. Here, we report the first structural study of mammalian Grh/CP2 factors. Crystal structures of the DNA-binding domains of grainyhead-like (Grhl) 1 and Grhl2 reveal a closely similar conformation with immunoglobulin-like core. Both share a common fold with the tumor suppressor p53, but differ in important structural features. The Grhl1 DNA-binding domain binds duplex DNA containing the consensus recognition element in a dimeric arrangement, supporting parsimonious target-sequence selection through two conserved arginine residues. We elucidate the molecular basis of a cancer-related mutation in Grhl1 involving one of these arginines, which completely abrogates DNA binding in biochemical assays and transcriptional activation of a reporter gene in a human cell line. Thus, our studies establish the structural basis of DNA target-site recognition by Grh transcription factors and reveal how tumor-associated mutations inactivate Grhl proteins. They may serve as points of departure for the structure-based development of Grh/CP2 inhibitors for therapeutic applications. PMID:29309642

A structural portrait of the PDZ domain family.

PubMed

Ernst, Andreas; Appleton, Brent A; Ivarsson, Ylva; Zhang, Yingnan; Gfeller, David; Wiesmann, Christian; Sidhu, Sachdev S

2014-10-23

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family. Copyright © 2014 Elsevier Ltd. All rights reserved.

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A.

PubMed

Grover, Abhinav; Agrawal, Vibhuti; Shandilya, Ashutosh; Bisaria, Virendra S; Sundar, Durai

2011-01-01

Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.

Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A

PubMed Central

2011-01-01

Background Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug. Results Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured. Conclusions We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus. PMID:22373101

GPR17: molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors.

PubMed

Parravicini, Chiara; Ranghino, Graziella; Abbracchio, Maria P; Fantucci, Piercarlo

2008-06-04

GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor. Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found. Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way.

Structural elucidation of estrus urinary lipocalin protein (EULP) and evaluating binding affinity with pheromones using molecular docking and fluorescence study

PubMed Central

Rajesh, Durairaj; Muthukumar, Subramanian; Saibaba, Ganesan; Siva, Durairaj; Akbarsha, Mohammad Abdulkader; Gulyás, Balázs; Padmanabhan, Parasuraman; Archunan, Govindaraju

2016-01-01

Transportation of pheromones bound with carrier proteins belonging to lipocalin superfamily is known to prolong chemo-signal communication between individuals belonging to the same species. Members of lipocalin family (MLF) proteins have three structurally conserved motifs for delivery of hydrophobic molecules to the specific recognizer. However, computational analyses are critically required to validate and emphasize the sequence and structural annotation of MLF. This study focused to elucidate the evolution, structural documentation, stability and binding efficiency of estrus urinary lipocalin protein (EULP) with endogenous pheromones adopting in-silico and fluorescence study. The results revealed that: (i) EULP perhaps originated from fatty acid binding protein (FABP) revealed in evolutionary analysis; (ii) Dynamic simulation study shows that EULP is highly stable at below 0.45 Å of root mean square deviation (RMSD); (iii) Docking evaluation shows that EULP has higher binding energy with farnesol and 2-iso-butyl-3-methoxypyrazine (IBMP) than 2-naphthol; and (iv) Competitive binding and quenching assay revealed that purified EULP has good binding interaction with farnesol. Both, In-silico and experimental studies showed that EULP is an efficient binding partner to pheromones. The present study provides impetus to create a point mutation for increasing longevity of EULP to develop pheromone trap for rodent pest management. PMID:27782155

Structural elucidation of estrus urinary lipocalin protein (EULP) and evaluating binding affinity with pheromones using molecular docking and fluorescence study.

PubMed

Rajesh, Durairaj; Muthukumar, Subramanian; Saibaba, Ganesan; Siva, Durairaj; Akbarsha, Mohammad Abdulkader; Gulyás, Balázs; Padmanabhan, Parasuraman; Archunan, Govindaraju

2016-10-26

Transportation of pheromones bound with carrier proteins belonging to lipocalin superfamily is known to prolong chemo-signal communication between individuals belonging to the same species. Members of lipocalin family (MLF) proteins have three structurally conserved motifs for delivery of hydrophobic molecules to the specific recognizer. However, computational analyses are critically required to validate and emphasize the sequence and structural annotation of MLF. This study focused to elucidate the evolution, structural documentation, stability and binding efficiency of estrus urinary lipocalin protein (EULP) with endogenous pheromones adopting in-silico and fluorescence study. The results revealed that: (i) EULP perhaps originated from fatty acid binding protein (FABP) revealed in evolutionary analysis; (ii) Dynamic simulation study shows that EULP is highly stable at below 0.45 Å of root mean square deviation (RMSD); (iii) Docking evaluation shows that EULP has higher binding energy with farnesol and 2-iso-butyl-3-methoxypyrazine (IBMP) than 2-naphthol; and (iv) Competitive binding and quenching assay revealed that purified EULP has good binding interaction with farnesol. Both, In-silico and experimental studies showed that EULP is an efficient binding partner to pheromones. The present study provides impetus to create a point mutation for increasing longevity of EULP to develop pheromone trap for rodent pest management.

Influence of the Selectivity Filter Properties on Proton Selectivity in the Influenza A M2 Channel.

PubMed

Dudev, Todor; Grauffel, Cédric; Lim, Carmay

2016-10-05

The homotetrameric M2 proton channel of influenza A plays a crucial role in the viral life cycle and is thus an important therapeutic target. It selectively conducts protons against a background of other competing cations whose concentrations are up to a million times greater than the proton concentration. Its selectivity is largely determined by a constricted region of its open pore known as the selectivity filter, which is lined by four absolutely conserved histidines. While the mechanism of proton transport through the channel has been studied, the physical principles underlying the selectivity for protons over other cations in the channel's His 4 selectivity filter remain elusive. Furthermore, it is not known if proton selectivity absolutely requires all four histidines with two of the four histidines protonated and if other titratable amino acid residues in lieu of the histidines could bind protons and how they affect proton selectivity. Here, we elucidate how the competition between protons and rival cations such as Na + depends on the selectivity filter's (1) histidine protonation state, (2) solvent exposure, (3) oligomeric state (the number of protein chains and thus the number of His ligands), and (4) ligand composition by evaluating the free energies for replacing monovalent Na + with H 3 O + in various model selectivity filters. We show that tetrameric His 4 filters are more proton-selective than their trimeric His 3 counterparts, and a dicationic His 4 filter where two of the four histidines are protonated is more proton-selective than tetrameric filters with other charge states/composition (different combinations of His protonation states or different metal-ligating ligands). The [His 4 ] 2+ filter achieves proton selectivity by providing suboptimal binding conditions for rival cations such as Na + , which prefers a neutral or negatively charged filter instead of a dicationic one, and three rather than four ligands with oxygen-ligating atoms.

Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence.

PubMed

Rawel, Harshadrai M; Frey, Simone K; Meidtner, Karina; Kroll, Jürgen; Schweigert, Florian J

2006-08-01

The noncovalent binding of selected phenolic compounds (chlorogenic-, ferulic-, gallic acid, quercetin, rutin, and isoquercetin) to proteins (HSA, BSA, soy glycinin, and lysozyme) was studied by an indirect method applying the quenching of intrinsic tryptophan fluorescence. From the data obtained, the binding constants were calculated by nonlinear regression (one site binding; y = Bx/k + x). It has been reported that tannins inhibit human salivary amylase and that these complexes may reduce the development of cariogenic plaques. Further, amylase contains two tryptophan residues in its active site. Therefore, in a second part of the study involving 31 human subjects, evidence was sought for noncovalent interactions between the phenols of green tea and saliva proteins as measured by the fluorescence intensity. Amylase activity was determined before and after the addition of green tea to saliva of 31 subjects. Forty percent of the subjects showed an increase in amylase activity contrary to studies reporting only a decrease in activity. The interactions of tannin with amylase result in a decrease of its activity. It still remains to be elucidated why amylase does not react uniformly under conditions of applying green tea to saliva. Further, in terms of using phenols as caries inhibitors this finding should be of importance.

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

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

Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan

2010-11-22

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues thatmore » flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.« less

Murine glomerulotropic monoclonal antibodies are highly oligoclonal and exhibit distinctive molecular features.

PubMed

Lefkowith, J B; Di Valerio, R; Norris, J; Glick, G D; Alexander, A L; Jackson, L; Gilkeson, G S

1996-08-01

We recently produced a panel of seven glomerular-binding mAbs from a nephritic MRL-lpr mouse that bind to histones/nucleosomes (group I) or DNA (group II) adherent to glomerular basement membrane. To elucidate the molecular basis of their binding and ontogeny, we sequenced their variable (V) regions, analyzed the apparent somatic mutations, and predicted their three-dimensional structures. There were two clonally related sets (3 of 4 in group I, 3 of 3 in group II) both of the VHJ1558 family, and one mAb of the VH 7183 family. V region somatic mutations within clonally related sets had little effect on glomerular binding and did not appear to be selected for based on glomerular binding. The VH regions were most homologous with those from autoantibodies to histones, DNA, or IgG (i.e., rheumatoid factors), the Vkappa regions, with those from autoantibodies to small nuclear ribonucleoproteins (snRNP). The VH regions also exhibited an unusual VD junction (in the group I clonally related set) and an overall high content of charged amino acids (arginine, aspartic acid) in complementarity-determining regions (CDRs), particularly in CDR3. Molecular modeling studies suggested that the Fv regions of these mAbs converge to form a flat, open surface with a net positive charge. The CDR arginines in group I mAbs; appear to be located in Ag contact regions of the binding cleft. In sum, these data suggest that glomerulotropic mAbs are a highly restricted set of Abs with distinctive molecular features that may mediate their binding to glomeruli.

Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

DOE PAGES

Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; ...

2016-09-01

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecularmore » interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. Finally, the in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.« less

Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1

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

Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong

DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecularmore » interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. Finally, the in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues.« less

Rate Constants and Mechanisms of Protein–Ligand Binding

PubMed Central

Pang, Xiaodong; Zhou, Huan-Xiang

2017-01-01

Whereas protein–ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms. PMID:28375732

Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine.

PubMed

Nakano, Hirofumi; Omura, Satoshi

2009-01-01

Staurosporine was discovered at the Kitasato Institute in 1977 while screening for microbial alkaloids using chemical detection methods. It was during the same era that protein kinase C was discovered and oncogene v-src was shown to have protein kinase activity. Staurosporine was first isolated from a culture of Actinomyces that originated in a soil sample collected in Mizusawa City, Japan. Thereafter, indolocarbazole compounds have been isolated from a variety of organisms. The biosynthesis of staurosporine and related indolocarbazoles was finally elucidated during the past decade through genetic and biochemical studies. Subsequently, several novel indolocarbazoles have been produced using combinatorial biosynthesis. In 1986, 9 years since its discovery, staurosporine and related indolocarbazoles were shown to be nanomolar inhibitors of protein kinases. They can thus be viewed as forerunners of today's crop of novel anticancer drugs. The finding led many pharmaceutical companies to search for selective protein kinase inhibitors by screening natural products and through chemical synthesis. In the 1990s, imatinib, a Bcr-Abl tyrosine kinase inhibitor, was synthesized and, following human clinical trials for chronic myelogenous leukemia, it was approved for use in the USA in 2001. In 1992, mammalian topoisomerases were shown to be targets for indolocarbazoles. This opened up new possibilities in that indolocarbazole compounds could selectively interact with ATP-binding sites of not only protein kinases but also other proteins that had slight differences in ATP-binding sites. ABCG2, an ATP-binding cassette transporter, was recently identified as an important new target for indolocarbazoles.

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

PubMed

Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

2017-11-20

For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase

PubMed Central

Ahmad, Zulfiqar; Hassan, Sherif S.; Azim, Sofiya

2017-01-01

For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phy-tochemicals is based on tradition or word of mouth with few evidence-based studies. Moreo-ver, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become perti-nent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of die-tary phytochemicals are known to inhibit ATP synthase. Structural modifications of phyto-chemicals have been shown to increase the inhibitory potency and extent of inhibition. Site-directed mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can re-sult in selective binding and inhibition of microbial ATP synthase. In this review, the therapeu-tic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective target-ing of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. PMID:28831918

Elucidating compound mechanism of action by network perturbation analysis | Office of Cancer Genomics

Cancer.gov

Genome-wide identification of the mechanism of action (MoA) of small-molecule compounds characterizing their targets, effectors, and activity modulators represents a highly relevant yet elusive goal, with critical implications for assessment of compound efficacy and toxicity. Current approaches are labor intensive and mostly limited to elucidating high-affinity binding target proteins.

Proteomic Analysis of an α7 Nicotinic Acetylcholine Receptor Interactome

PubMed Central

Paulo, Joao A.; Brucker, William J.; Hawrot, Edward

2009-01-01

The α7 nicotinic acetylcholine receptor (nAChR) is well established as the principal high-affinity α-bungarotoxin-binding protein in the mammalian brain. We isolated carbachol-sensitive α-bungarotoxin-binding complexes from total mouse brain tissue by affinity immobilization followed by selective elution, and these proteins were fractionated by SDS-PAGE. The proteins in subdivided gel lane segments were tryptically digested, and the resulting peptides were analyzed by standard mass spectrometry. We identified 55 proteins in wild-type samples that were not present in comparable brain samples from α7 nAChR knockout mice that had been processed in a parallel fashion. Many of these 55 proteins are novel proteomic candidates for interaction partners of the α7 nAChR, and many are associated with multiple signaling pathways that may be implicated in α7 function in the central nervous system. The newly identified potential protein interactions, together with the general methodology that we introduce for α-bungarotoxin-binding protein complexes, form a new platform for many interesting follow-up studies aimed at elucidating the physiological role of neuronal α7 nAChRs. PMID:19714875

Imaging dopamine D3 receptors in the human brain with positron emission tomography, [11C]PHNO, and a selective D3 receptor antagonist.

PubMed

Searle, Graham; Beaver, John D; Comley, Robert A; Bani, Massimo; Tziortzi, Andri; Slifstein, Mark; Mugnaini, Manolo; Griffante, Cristiana; Wilson, Alan A; Merlo-Pich, Emilio; Houle, Sylvain; Gunn, Roger; Rabiner, Eugenii A; Laruelle, Marc

2010-08-15

Dopamine D(3) receptors are involved in the pathophysiology of several neuropsychiatric conditions. [(11)C]-(+)-PHNO is a radiolabeled D(2) and D(3) agonist, suitable for imaging the agonist binding sites (denoted D(2HIGH) and D(3)) of these receptors with positron emission tomography (PET). PET studies in nonhuman primates documented that, in vivo, [(11)C]-(+)-PHNO displays a relative selectivity for D(3) compared with D(2HIGH) receptor sites and that the [(11)C]-(+)-PHNO signal is enriched in D(3) contribution compared with conventional ligands such as [(11)C] raclopride. To define the D(3) contribution (f(PHNO)(D3)) to [(11)C]-(+)-PHNO binding potential (BP(ND)) in healthy humans, 52 PET scans were obtained in 19 healthy volunteers at baseline and following oral administration of various doses of the selective D(3) receptor antagonist, GSK598809. The impact of GSK598809 on [(11)C]-(+)-PHNO was regionally selective. In dorsal regions of the striatum, GSK598809 did not significantly affect [(11)C]-(+)-PHNO BP(ND) (f(PHNO)(D3) approximately 0%). Conversely, in the substantia nigra, GSK598809 dose-dependently reduced [(11)C]-(+)-PHNO binding to nonspecific level (f(PHNO)(D3) approximately 100%). In ventral striatum (VST), globus pallidus and thalamus (THA), [(11)C]-(+)-PHNO BP(ND) was attributable to a combination of D(2HIGH) and D(3) receptor sites, with f(PHNO)(D3) of 26%, 67% and 46%, respectively. D(3) receptor binding potential (BP(ND)(D3)) was highest in globus pallidus (1.90) and substantial nigra (1.39), with lower levels in VST (.77) and THA (.18) and negligible levels in dorsal striatum. This study elucidated the pharmacologic nature of the [(11)C]-(+)-PHNO signal in healthy subjects and provided the first quantification of D(3) receptor availability with PET in the living human brain. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

A designed inhibitor of a CLC antiporter blocks function through a unique binding mode

PubMed Central

Howery, Andrew E.; Elvington, Shelley; Abraham, Sherwin J.; Choi, Kee-Hyun; Phillips, Sabrina; Ryan, Christopher M.; Sanford, R. Lea; Simpson-Dworschak, Sierra; Almqvist, Jonas; Tran, Kevin; Chew, Thomas A.; Zachariae, Ulrich; Andersen, Olaf S.; Whitelegge, Julian; Matulef, Kimberly; Du Bois, Justin; Maduke, Merritt C.

2012-01-01

SUMMARY The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC “Chloride Channel” family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of the first specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4′-octanamidostilbene-2,2′-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid-dependence of inhibition suggest potential mechanisms of action. The discovery of this compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders. PMID:23177200

Inhibition of protein tyrosine phosphatase 1B as a potential treatment of diabetes and obesity.

PubMed

Pei, Zhonghua; Liu, Gang; Lubben, Thomas H; Szczepankiewicz, Bruce G

2004-01-01

Diabetes is a prevalent disease which effects over 150 million people worldwide and there is a great medical need for new therapeutic agents to treat it. Inhibition of protein tyrosine phosphatase 1B (PTP1B) has emerged as a highly validated, attractive target for treatment of not only diabetes but also obesity. Discovery of small-molecule inhibitors has been pursued extensively in both academia and industry and a number of very potent and selective inhibitors have been identified. With X-ray crystallography, the binding interactions of several classes of inhibitors have been elucidated. This has resulted in significant progress in understanding important interactions between inhibitors and specific residues of PTP1B, which could help the design of future inhibitors. However, since the active site of PTP1B that most of these inhibitors bind to is highly hydrophilic, it remains a challenge to identify inhibitors with both excellent in vitro potency and drug-like physiochemical properties which would lead to good in vivo activities.

High performance wash-free magnetic bioassays through microfluidically enhanced particle specificity.

PubMed

Bechstein, Daniel J B; Lee, Jung-Rok; Ooi, Chin Chun; Gani, Adi W; Kim, Kyunglok; Wilson, Robert J; Wang, Shan X

2015-06-30

Magnetic biosensors have emerged as a sensitive and versatile platform for high performance medical diagnostics. These magnetic biosensors require well-tailored magnetic particles as detection probes, which need to give rise to a large and specific biological signal while showing very low nonspecific binding. This is especially important in wash-free bioassay protocols, which do not require removal of particles before measurement, often a necessity in point of care diagnostics. Here we show that magnetic interactions between magnetic particles and magnetized sensors dramatically impact particle transport and magnetic adhesion to the sensor surfaces. We investigate the dynamics of magnetic particles' biomolecular binding and magnetic adhesion to the sensor surface using microfluidic experiments. We elucidate how flow forces can inhibit magnetic adhesion, greatly diminishing or even eliminating nonspecific signals in wash-free magnetic bioassays, and enhancing signal to noise ratios by several orders of magnitude. Our method is useful for selecting and optimizing magnetic particles for a wide range of magnetic sensor platforms.

High performance wash-free magnetic bioassays through microfluidically enhanced particle specificity

PubMed Central

Bechstein, Daniel J.B.; Lee, Jung-Rok; Ooi, Chin Chun; Gani, Adi W.; Kim, Kyunglok; Wilson, Robert J.; Wang, Shan X.

2015-01-01

Magnetic biosensors have emerged as a sensitive and versatile platform for high performance medical diagnostics. These magnetic biosensors require well-tailored magnetic particles as detection probes, which need to give rise to a large and specific biological signal while showing very low nonspecific binding. This is especially important in wash-free bioassay protocols, which do not require removal of particles before measurement, often a necessity in point of care diagnostics. Here we show that magnetic interactions between magnetic particles and magnetized sensors dramatically impact particle transport and magnetic adhesion to the sensor surfaces. We investigate the dynamics of magnetic particles’ biomolecular binding and magnetic adhesion to the sensor surface using microfluidic experiments. We elucidate how flow forces can inhibit magnetic adhesion, greatly diminishing or even eliminating nonspecific signals in wash-free magnetic bioassays, and enhancing signal to noise ratios by several orders of magnitude. Our method is useful for selecting and optimizing magnetic particles for a wide range of magnetic sensor platforms. PMID:26123868

The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer.

PubMed

Yu, Sungryul; Kim, Taemook; Yoo, Kyung Hyun; Kang, Keunsoo

2017-05-06

Cell lines are often used as in vitro tools to mimic certain types of in vivo system; several cell lines, including MCF-7 and T47D, have been widely used in breast cancer studies without investigating the cell lines' characteristics. In this study, we compared the genome-wide binding profiles of ERα, PR, and P300, and the gene expression changes between MCF-7 and T47D cell lines that represent the luminal A subtype of breast cancer. Surprisingly, several thousand genes were differentially expressed under estrogenic condition. In addition, ERα, PR, and P300 binding to regulatory elements showed distinct genomic landscapes between MCF-7 and T47D cell lines in the same hormonal states. In particular, the T47D cell line was markedly susceptible to progesterone, whereas the MCF-7 cell line did not respond to progesterone in the presence of estrogen. Consistently, changes in the expression level of the PR-target gene, STAT5A, were only observed in the T47D cell line, not the MCF-7 cell line, when treated with progesterone. Overall, the results highlight the importance of selecting appropriate cell lines for breast cancer studies and suggest that T47D cell lines can be an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering Visual Arrestin-1 with Special Functional Characteristics*

PubMed Central

Vishnivetskiy, Sergey A.; Chen, Qiuyan; Palazzo, Maria C.; Brooks, Evan K.; Altenbach, Christian; Iverson, Tina M.; Hubbell, Wayne L.; Gurevich, Vsevolod V.

2013-01-01

Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations. PMID:23250748

Voltage-gated sodium channels as targets for pyrethroid insecticides.

PubMed

Field, Linda M; Emyr Davies, T G; O'Reilly, Andrias O; Williamson, Martin S; Wallace, B A

2017-10-01

The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity.

ATP-induced conformational changes of nucleotide-binding domains in an ABC transporter. Importance of the water-mediated entropic force.

PubMed

Hayashi, Tomohiko; Chiba, Shuntaro; Kaneta, Yusuke; Furuta, Tadaomi; Sakurai, Minoru

2014-11-06

ATP binding cassette (ABC) proteins belong to a superfamily of active transporters. Recent experimental and computational studies have shown that binding of ATP to the nucleotide binding domains (NBDs) of ABC proteins drives the dimerization of NBDs, which, in turn, causes large conformational changes within the transmembrane domains (TMDs). To elucidate the active substrate transport mechanism of ABC proteins, it is first necessary to understand how the NBD dimerization is driven by ATP binding. In this study, we selected MalKs (NBDs of a maltose transporter) as a representative NBD and calculated the free-energy change upon dimerization using molecular mechanics calculations combined with a statistical thermodynamic theory of liquids, as well as a method to calculate the translational, rotational, and vibrational entropy change. This combined method is applied to a large number of snapshot structures obtained from molecular dynamics simulations containing explicit water molecules. The results suggest that the NBD dimerization proceeds with a large gain of water entropy when ATP molecules bind to the NBDs. The energetic gain arising from direct NBD-NBD interactions is canceled by the dehydration penalty and the configurational-entropy loss. ATP hydrolysis induces a loss of the shape complementarity between the NBDs, which leads to the dissociation of the dimer, due to a decrease in the water-entropy gain and an increase in the configurational-entropy loss. This interpretation of the NBD dimerization mechanism in concert with ATP, especially focused on the water-mediated entropy force, is potentially applicable to a wide variety of the ABC transporters.

Locating the route of entry and binding sites of benzocaine and phenytoin in a bacterial voltage gated sodium channel.

PubMed

Martin, Lewis J; Corry, Ben

2014-07-01

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

Locating the Route of Entry and Binding Sites of Benzocaine and Phenytoin in a Bacterial Voltage Gated Sodium Channel

PubMed Central

Martin, Lewis J.; Corry, Ben

2014-01-01

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites. PMID:24992293

A molecular dynamics investigation into the mechanisms of alectinib resistance of three ALK mutants.

PubMed

He, Muyang; Li, Weikang; Zheng, Qingchuan; Zhang, Hongxing

2018-07-01

Alectinib, a highly selective next-genetation anaplastic lymphoma kinase (ALK) inhibitor, has demonstrated promising antitumor activity in patients with ALK-positive non-small cell lung carcinomas (NSCLC). However, the therapeutic benefits of alectinib is inescapably hampered by the development of acquired resistant mutations in ALK. Despite the availability of ample experimental mutagenesis data, the molecular origin and the structural motifs under alectinib binding affinity deficiencies are still ambiguous. Here, molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) calculation approaches were employed to elucidate the mechanisms of alectinib resistance induced by the mutations I1171N, V1180L, and L1198F. The MD results reveal that the studied mutations could trigger the dislocation of alectinib as well as conformational changes at the inhibitor binding site, thus induce the interactional changes between alectinib and mutants. The most influenced regions are the ligand binding entrance and the hinge region, which are considered to be the dominant binding motifs accounting for the binding affinity loss in mutants. The "key and lock mechanism" between the ethyl group at position 9 of alectinib and a recognition cavity in the hinge region of ALK is presented to illustrate the major molecular origin of drug resistance. Our results provide mechanistic insight into the effect of ALK mutations resistant to alectinib, which could contribute to further rational design of inhibitors to combat the acquired resistance. © 2018 Wiley Periodicals, Inc.

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity.

PubMed

Iwanowicz, Edwin J; Kimball, S David; Lin, James; Lau, Wan; Han, W-C; Wang, Tammy C; Roberts, Daniel G M; Schumacher, W A; Ogletree, Martin L; Seiler, Steven M

2002-11-04

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.

Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

PubMed

Istyastono, Enade P; Nijmeijer, Saskia; Lim, Herman D; van de Stolpe, Andrea; Roumen, Luc; Kooistra, Albert J; Vischer, Henry F; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

2011-12-08

The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

Plasma Levels of Fatty Acid-Binding Protein 4, Retinol-Binding Protein 4, High-Molecular-Weight Adiponectin, and Cardiovascular Mortality Among Men With Type 2 Diabetes: A 22-Year Prospective Study.

PubMed

Liu, Gang; Ding, Ming; Chiuve, Stephanie E; Rimm, Eric B; Franks, Paul W; Meigs, James B; Hu, Frank B; Sun, Qi

2016-11-01

To examine select adipokines, including fatty acid-binding protein 4, retinol-binding protein 4, and high-molecular-weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes mellitus. Plasma levels of fatty acid-binding protein 4, retinol-binding protein 4, and HMW adiponectin were measured in 950 men with type 2 diabetes mellitus in the Health Professionals Follow-up Study. After an average of 22 years of follow-up (1993-2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of fatty acid-binding protein 4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio and 95% confidence interval of CVD mortality was 1.78 (1.22-2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the hazard ratio (95% confidence interval) was 2.07 (1.42-3.06; P trend=0.0002), comparing extreme tertiles, whereas higher retinol-binding protein 4 levels were nonsignificantly associated with a decreased CVD mortality with an hazard ratio (95% confidence interval) of 0.73 (0.50-1.07; P trend=0.09). A Mendelian randomization analysis suggested that the causal relationships of HMW adiponectin and retinol-binding protein 4 would be directionally opposite to those observed based on the biomarkers, although none of the Mendelian randomization associations achieved statistical significance. These data suggest that higher levels of fatty acid-binding protein 4 and HMW adiponectin are associated with elevated CVD mortality among men with type 2 diabetes mellitus. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among patients with type 2 diabetes mellitus. © 2016 American Heart Association, Inc.

Current Understanding of the Binding Sites, Capacity, Affinity, and Biological Significance of Metals in Melanin

PubMed Central

Hong, Lian; Simon, John D.

2008-01-01

Metal chelation is often invoked as one of the main biological functions of melanin. In order to understand the interaction between metals and melanin, extensive studies have been carried out to determine the nature of the metal binding sites, binding capacity and affinity. These data are central to efforts aimed at elucidating the role metal binding plays in determining the physical, structural, biological, and photochemical properties of melanin. This article examines the current state of understanding of this field. PMID:17580858

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

PubMed Central

Dixon-Clarke, Sarah E.; Shehata, Saifeldin N.; Krojer, Tobias; Sharpe, Timothy D.; vonDelft, Frank; Sakamoto, Kei

2017-01-01

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. PMID:28057719

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

PubMed Central

Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

2002-01-01

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

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.

Toxin studies using an integrated biophysical and structural biology approach.

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

Last, Julie A.; Schroeder, Anne E.; Slade, Andrea Lynn

2005-03-01

Clostridial neurotoxins, such as botulinum and tetanus, are generally thought to invade neural cells through a process of high affinity binding mediated by gangliosides, internalization via endosome formation, and subsequent membrane penetration of the catalytic domain activated by a pH drop in the endosome. This surface recognition and internalization process is still not well understood with regard to what specific membrane features the toxins target, the intermolecular interactions between bound toxins, and the molecular conformational changes that occur as a result of pH lowering. In an effort to elucidate the mechanism of tetanus toxin binding and permeation through the membranemore » a simple yet representative model was developed that consisted of the ganglioside G{sub tlb} incorporated in a bilayer of cholesterol and DPPC (dipalmitoylphosphatidyl choline). The bilayers were stable over time yet sensitive towards the binding and activity of whole toxin. A liposome leakage study at constant pH as well as with a pH gradient, to mimic the processes of the endosome, was used to elucidate the effect of pH on the toxin's membrane binding and permeation capability. Topographic imaging of the membrane surface, via in situ tapping mode AFM, provided nanoscale characterization of the toxin's binding location and pore formation activity.« less

First-second shell interactions in metal binding sites in proteins: a PDB survey and DFT/CDM calculations.

PubMed

Dudev, Todor; Lin, Yen-lin; Dudev, Minko; Lim, Carmay

2003-03-12

The role of the second shell in the process of metal binding and selectivity in metalloproteins has been elucidated by combining Protein Data Bank (PDB) surveys of Mg, Mn, Ca, and Zn binding sites with density functional theory/continuum dielectric methods (DFT/CDM). Peptide backbone groups were found to be the most common second-shell ligand in Mg, Mn, Ca, and Zn binding sites, followed (in decreasing order) by Asp/Glu, Lys/Arg, Asn/Gln, and Ser/Thr side chains. Aromatic oxygen- or nitrogen-containing side chains (Tyr, His, and Trp) and sulfur-containing side chains (Cys and Met) are seldom found in the second coordination layer. The backbone and Asn/Gln side chain are ubiquitous in the metal second coordination layer as their carbonyl oxygen and amide hydrogen can act as a hydrogen-bond acceptor and donor, respectively, and can therefore partner practically every first-shell ligand. The second most common outer-shell ligand, Asp/Glu, predominantly hydrogen bonds to a metal-bound water or Zn-bound histidine and polarizes the H-O or H-N bond. In certain cases, a second-shell Asp/Glu could affect the protonation state of the metal ligand. It could also energetically stabilize a positively charged metal complex more than a neutral ligand such as the backbone and Asn/Gln side chain. As for the first shell, the second shell is predicted to contribute to the metal selectivity of the binding site by discriminating between metal cations of different ionic radii and coordination geometries. The first-shell-second-shell interaction energies decay rapidly with increasing solvent exposure of the metal binding site. They are less favorable but are of the same order of magnitude as compared to the respective metal-first-shell interaction energies. Altogether, the results indicate that the structure and properties of the second shell are dictated by those of the first layer. The outer shell is apparently designed to stabilize/protect the inner-shell and complement/enhance its properties.

Genome analysis and signature discovery for diving and sensory properties of the endangered Chinese alligator

PubMed Central

Wan, Qiu-Hong; Pan, Sheng-Kai; Hu, Li; Zhu, Ying; Xu, Peng-Wei; Xia, Jin-Quan; Chen, Hui; He, Gen-Yun; He, Jing; Ni, Xiao-Wei; Hou, Hao-Long; Liao, Sheng-Guang; Yang, Hai-Qiong; Chen, Ying; Gao, Shu-Kun; Ge, Yun-Fa; Cao, Chang-Chang; Li, Peng-Fei; Fang, Li-Ming; Liao, Li; Zhang, Shu; Wang, Meng-Zhen; Dong, Wei; Fang, Sheng-Guo

2013-01-01

Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tibetan Plateau was uplifted. PMID:23917531

Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

PubMed

Brucet, Marina; Querol-Audí, Jordi; Serra, Maria; Ramirez-Espain, Ximena; Bertlik, Kamila; Ruiz, Lidia; Lloberas, Jorge; Macias, Maria J; Fita, Ignacio; Celada, Antonio

2007-05-11

TREX1 is the most abundant mammalian 3' --> 5' DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

Computational insight into small molecule inhibition of cyclophilins.

PubMed

Sambasivarao, Somisetti V; Acevedo, Orlando

2011-02-28

Cyclophilins (Cyp) are a family of cellular enzymes possessing peptidyl-prolyl isomerase activity, which catalyze the cis-trans interconversion of proline-containing peptide bonds. The two most abundant family members, CypA and CypB, have been identified as valid drug targets for a wide range of diseases, including HCV, HIV, and multiple cancers. However, the development of small molecule inhibitors that possess nM potency and high specificity for a particular Cyp is difficult given the complete conservation of all active site residues between the enzymes. Monte Carlo statistical sampling coupled to free energy perturbation theory (MC/FEP) calculations have been carried out to elucidate the origin of the experimentally observed nM inhibition of CypA by acylurea-based derivatives and the >200-fold in vitro selectivity between CypA and CypB from aryl 1-indanylketone-based μM inhibitors. The computed free-energies of binding were in close accord with those derived from experiments. Binding affinity values for the inhibitors were determined to be dependent upon the stabilization strength of the nonbonded interactions provided toward two catalytic residues: Arg55 and Asn102 in CypA and the analogous Arg63 and Asn110 residues in CypB. Fine-tuning of the hydrophobic interactions allowed for enhanced potency among derivatives. The aryl 1-indanylketones are predicted to differentiate between the cyclophilins by using distinct binding motifs that exploit subtle differences in the active site arrangements. Ideas for the development of new selective compounds with the potential for advancement to low-nanomolar inhibition are presented.

Genome analysis and signature discovery for diving and sensory properties of the endangered Chinese alligator.

PubMed

Wan, Qiu-Hong; Pan, Sheng-Kai; Hu, Li; Zhu, Ying; Xu, Peng-Wei; Xia, Jin-Quan; Chen, Hui; He, Gen-Yun; He, Jing; Ni, Xiao-Wei; Hou, Hao-Long; Liao, Sheng-Guang; Yang, Hai-Qiong; Chen, Ying; Gao, Shu-Kun; Ge, Yun-Fa; Cao, Chang-Chang; Li, Peng-Fei; Fang, Li-Ming; Liao, Li; Zhang, Shu; Wang, Meng-Zhen; Dong, Wei; Fang, Sheng-Guo

2013-09-01

Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tibetan Plateau was uplifted.

In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function.

PubMed

Follin, Elna; Karlsson, Maria; Lundegaard, Claus; Nielsen, Morten; Wallin, Stefan; Paulsson, Kajsa; Westerdahl, Helena

2013-04-01

The major histocompatibility complex (MHC) genes are the most polymorphic genes found in the vertebrate genome, and they encode proteins that play an essential role in the adaptive immune response. Many songbirds (passerines) have been shown to have a large number of transcribed MHC class I genes compared to most mammals. To elucidate the reason for this large number of genes, we compared 14 MHC class I alleles (α1-α3 domains), from great reed warbler, house sparrow and tree sparrow, via phylogenetic analysis, homology modelling and in silico peptide-binding predictions to investigate their functional and genetic relationships. We found more pronounced clustering of the MHC class I allomorphs (allele specific proteins) in regards to their function (peptide-binding specificities) compared to their genetic relationships (amino acid sequences), indicating that the high number of alleles is of functional significance. The MHC class I allomorphs from house sparrow and tree sparrow, species that diverged 10 million years ago (MYA), had overlapping peptide-binding specificities, and these similarities across species were also confirmed in phylogenetic analyses based on amino acid sequences. Notably, there were also overlapping peptide-binding specificities in the allomorphs from house sparrow and great reed warbler, although these species diverged 30 MYA. This overlap was not found in a tree based on amino acid sequences. Our interpretation is that convergent evolution on the level of the protein function, possibly driven by selection from shared pathogens, has resulted in allomorphs with similar peptide-binding repertoires, although trans-species evolution in combination with gene conversion cannot be ruled out.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein.

PubMed

Chao, Kinlin L; Kulakova, Liudmila; Herzberg, Osnat

2017-02-14

The exact function of human gasdermin-B (GSDMB), which regulates differentiation and growth of epithelial cells, is yet to be elucidated. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, GSDMB gene amplification and protein overexpression indicate a poor response to HER2-targeted therapy. Genome-wide association studies revealed a correlation between GSDMB SNPs and an increased susceptibility to Crohn's disease, ulcerative colitis, and asthma. The N- and C-terminal domains of all gasdermins possess lipid-binding and regulatory activities, respectively. Inflammatory caspases cleave gasdermin-D in the interdomain linker but not GSDMB. The cleaved N-terminal domain binds phosphoinositides and cardiolipin, forms membrane-disrupting pores, and executes pyroptosis. We show that both full-length GSDMB and the N-terminal domain bind to nitrocellulose membranes immobilized with phosphoinositides or sulfatide, but not with cardiolipin. In addition, the GSDMB N-terminal domain binds liposomes containing sulfatide. The crystal structure of the GSDMB C-terminal domain reveals the structural impact of the amino acids encoded by SNPs that are linked to asthma and inflammatory bowel disease (IBD). A loop that carries the polymorphism amino acids corresponding to healthy individuals (Gly299:Pro306) exhibits high conformational flexibility, whereas the loop carrying amino acids found in individuals with increased disease risk (Arg299:Ser306) exhibits a well-defined conformation and higher positive surface charge. Apoptotic executioner caspase-3, -6, and -7, but not the inflammatory caspases, cleave GSDMB at 88 DNVD 91 within the N-terminal domain. Selective sulfatide binding may indicate possible function for GSDMB in the cellular sulfatide transport.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein

PubMed Central

Chao, Kinlin L.; Kulakova, Liudmila; Herzberg, Osnat

2017-01-01

The exact function of human gasdermin-B (GSDMB), which regulates differentiation and growth of epithelial cells, is yet to be elucidated. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, GSDMB gene amplification and protein overexpression indicate a poor response to HER2-targeted therapy. Genome-wide association studies revealed a correlation between GSDMB SNPs and an increased susceptibility to Crohn’s disease, ulcerative colitis, and asthma. The N- and C-terminal domains of all gasdermins possess lipid-binding and regulatory activities, respectively. Inflammatory caspases cleave gasdermin-D in the interdomain linker but not GSDMB. The cleaved N-terminal domain binds phosphoinositides and cardiolipin, forms membrane-disrupting pores, and executes pyroptosis. We show that both full-length GSDMB and the N-terminal domain bind to nitrocellulose membranes immobilized with phosphoinositides or sulfatide, but not with cardiolipin. In addition, the GSDMB N-terminal domain binds liposomes containing sulfatide. The crystal structure of the GSDMB C-terminal domain reveals the structural impact of the amino acids encoded by SNPs that are linked to asthma and inflammatory bowel disease (IBD). A loop that carries the polymorphism amino acids corresponding to healthy individuals (Gly299:Pro306) exhibits high conformational flexibility, whereas the loop carrying amino acids found in individuals with increased disease risk (Arg299:Ser306) exhibits a well-defined conformation and higher positive surface charge. Apoptotic executioner caspase-3, -6, and -7, but not the inflammatory caspases, cleave GSDMB at 88DNVD91 within the N-terminal domain. Selective sulfatide binding may indicate possible function for GSDMB in the cellular sulfatide transport. PMID:28154144

Applications of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) to study interactions of genetically engineered proteins with noble metal films

NASA Astrophysics Data System (ADS)

Suzuki, Noriaki

Genetically engineered proteins for inorganics (GEPIs) belong to a new class of polypeptides that are designed to have specific affinities to inorganic materials. A "gold binding protein (GBP)" was chosen as a model protein for GEPIs to study the molecular origins of binding specificity to gold using Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS). TOF-SIMS, a surface-sensitive analytical instrument with extremely high mass resolutions, provides information on specific amino acid-surface interactions. We used "principal component analysis (PCA)" to analyze the data. We also introduced a new multivariate technique, "hierarchical cluster analysis (HCA)" to organize the data into meaningful structures by measuring a degree of "similarity" and "dissimilarity" of the data. This report discusses a combined use of PCA and HCA to elucidate the binding specificity of GBP to Au. Based on the knowledge gained from TOF-SIMS measurements, we further investigated the nature of the interaction between selected amino acids and noble metal surfaces by using X-ray photoelectron spectroscopy (XPS). We developed a unique capability to introduce water vapor during the adsorption of a single amino acid and applied this method to study the intrinsic nature of sidechain/Au interactions. To further apply this unique research protocol, we characterized another type of GEPI, "quartz binding protein (QBP)," to identify the possible binding sites. This thesis research aims to provide experimental protocols for analyzing short peptide-substrate interface from complex spectroscopic data by using multivariate analysis techniques.

Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-3α.

PubMed

Lo Monte, Fabio; Kramer, Thomas; Gu, Jiamin; Anumala, Upendra Rao; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Franco, Bénédicte; Demedts, David; Van Leuven, Fred; Fuertes, Ana; Dominguez, Juan Manuel; Plotkin, Batya; Eldar-Finkelman, Hagit; Schmidt, Boris

2012-05-10

The glycogen synthase kinase-3 (GSK-3) has been linked to the pathogenesis of colorectal cancer, diabetes, cardiovascular disease, acute myeloid leukemia (AML), and Alzheimer's disease (AD). The debate on the respective contributions of GSK-3α and GSK-3β to AD pathology and AML is ongoing. Thus, the identification of potent GSK-3α-selective inhibitors, endowed with favorable pharmacokinetic properties, may elucidate the effect of GSK-3α inhibition in AD and AML models. The analysis of all available crystallized GSK-3 structures provided a simplified scheme of the relevant hot spots responsible for ligand binding and potency. This resulted in the identification of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest GSK-3α selectivity reported so far. In addition, compound 14d did not display significant inhibition of 48 out of 50 kinases in the test panel. The GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay.

Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes.

PubMed

Kokla, Anna; Blouchos, Petros; Livaniou, Evangelia; Zikos, Christos; Kakabakos, Sotiris E; Petrou, Panagiota S; Kintzios, Spyridon

2013-12-01

Membrane engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor-like molecules on the cell surface. Previous studies have elucidated the biochemical aspects of the interaction between various analytes (including viruses) and their homologous membrane-engineered cells. In the present study, purified anti-biotin antibodies from a rabbit antiserum along with in-house prepared biotinylated bovine serum albumin (BSA) were used as a model antibody-antigen pair of molecules for facilitating membrane engineering experiments. It was proven, with the aid of fluorescence microscopy, that (i) membrane-engineered cells incorporated the specific antibodies in the correct orientation and that (ii) the inserted antibodies are selectively interacting with the homologous target molecules. This is the first time the actual working concept of membrane engineering has been visualized, thus providing a final proof of the concept behind this innovative process. In addition, the fluorescence microscopy measurements were highly correlated with bioelectric measurements done with the aid of a bioelectric recognition assay. Copyright © 2013 John Wiley & Sons, Ltd.

Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

PubMed Central

Clerc, Jérôme; Groll, Michael; Illich, Damir J.; Bachmann, André S.; Huber, Robert; Schellenberg, Barbara; Dudler, Robert; Kaiser, Markus

2009-01-01

Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactin-based proteasome inhibitor described so far. With a Ki′ of 8.65 ± 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics. PMID:19359491

HIV-1 Envelope Glycoproteins from Diverse Clades Differentiate Antibody Responses and Durability among Vaccinees

PubMed Central

2018-01-01

ABSTRACT Induction of broadly cross-reactive antiviral humoral responses with the capacity to target globally diverse circulating strains is a key goal for HIV-1 immunogen design. A major gap in the field is the identification of diverse HIV-1 envelope antigens to evaluate vaccine regimens for binding antibody breadth. In this study, we define unique antigen panels to map HIV-1 vaccine-elicited antibody breadth and durability. Diverse HIV-1 envelope glycoproteins were selected based on genetic and geographic diversity to cover the global epidemic, with a focus on sexually acquired transmitted/founder viruses with a tier 2 neutralization phenotype. Unique antigenicity was determined by nonredundancy (Spearman correlation), and antigens were clustered using partitioning around medoids (PAM) to identify antigen diversity. Cross-validation demonstrated that the PAM method was better than selection by reactivity and random selection. Analysis of vaccine-elicited V1V2 binding antibody in longitudinal samples from the RV144 clinical trial revealed the striking heterogeneity among individual vaccinees in maintaining durable responses. These data support the idea that a major goal for vaccine development is to improve antibody levels, breadth, and durability at the population level. Elucidating the level and durability of vaccine-elicited binding antibody breadth needed for protection is critical for the development of a globally efficacious HIV vaccine. IMPORTANCE The path toward an efficacious HIV-1 vaccine will require characterization of vaccine-induced immunity that can recognize and target the highly genetically diverse virus envelope glycoproteins. Antibodies that target the envelope glycoproteins, including diverse sequences within the first and second hypervariable regions (V1V2) of gp120, were identified as correlates of risk for the one partially efficacious HIV-1 vaccine. To build upon this discovery, we experimentally and computationally evaluated humoral responses to define envelope glycoproteins representative of the antigenic diversity of HIV globally. These diverse envelope antigens distinguished binding antibody breadth and durability among vaccine candidates, thus providing insights for advancing the most promising HIV-1 vaccine candidates. PMID:29386288

Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

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

Joshi, Rakesh; Ho, Kwok Ki; Tenney, Kristen

2013-09-18

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{submore » +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.« less

NF-{kappa}B regulates Lef1 gene expression in chondrocytes

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

Yun, Kangsun; Choi, Yoo Duk; Nam, Jong Hee

The relation of Wnt/{beta}-catenin signaling to osteoarthritis progression has been revealed with little information on the underlying molecular mechanism. In this study we found overexpression of Lef1 in cartilage tissue of osteoarthritic patients and elucidated molecular mechanism of NF-{kappa}B-mediated Lef1 gene regulation in chondrocytes. Treatment of IL-1{beta} augmented Lef1 upregulation and nuclear translocation of NF-{kappa}B in chondrocytes. Under IL-1{beta} signaling, treatment of NF-{kappa}B nuclear translocation inhibitor SN-50 reduced Lef1 expression. A conserved NF-{kappa}B-binding site between mouse and human was selected through bioinformatic analysis and mapped at the 14 kb upstream of Lef1 transcription initiation site. NF-{kappa}B binding to the sitemore » was confirmed by chromatin immunoprecipitation assay. Lef1 expression was synergistically upregulated by interactions of NF-{kappa}B with Lef1/{beta}-catenin in chondrocytes. Our results suggest a pivotal role of NF-{kappa}B in Lef1 expression in arthritic chondrocytes or cartilage degeneration.« less

High-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination.

PubMed

Tan, Yann-Chong; Blum, Lisa K; Kongpachith, Sarah; Ju, Chia-Hsin; Cai, Xiaoyong; Lindstrom, Tamsin M; Sokolove, Jeremy; Robinson, William H

2014-03-01

We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination. Published by Elsevier Inc.

Molecular basis of ubiquitin recognition by the autophagy receptor CALCOCO2

PubMed Central

Xie, Xingqiao; Li, Faxiang; Wang, Yuanyuan; Wang, Yingli; Lin, Zhijie; Cheng, Xiaofang; Liu, Jianping; Chen, Changbin; Pan, Lifeng

2015-01-01

The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C2H2-type zinc-finger, and only the C2H2-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C2H2-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations. PMID:26506893

Translational regulation of inhibin βA by TGFβ via the RNA-binding protein hnRNP E1 enhances the invasiveness of epithelial-to-mesenchymal transitioned cells.

PubMed

Howley, B V; Hussey, G S; Link, L A; Howe, P H

2016-03-31

The epithelial-to-mesenchymal transition (EMT) is a cellular process that functions during embryonic development and tissue regeneration, thought to be aberrantly activated in epithelial-derived cancer and has an important role in the process of metastasis. The transforming growth factor (TGF)-β signaling pathway is a key inducer of EMT and we have elucidated a posttranscriptional mechanism by which TGFβ modulates expression of select transcripts via the RNA-binding protein hnRNP E1 during EMT. One such transcript inhibin βA is a member of the TGFβ superfamily. Here, we show by polysome profiling that inhibin βA is translationally regulated by TGFβ via hnRNP E1. TGFβ treatment or knockdown of hnRNP E1 relieves silencing of the inhibin βA transcript, resulting in increased protein expression and secreted levels of the inhibin βA homodimer, activin A. Our data indicate that the translational upregulation of inhibin βA enhances the migration and invasion of cells that have undergone an EMT and promotes cancer progression in vivo.

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

PubMed Central

Huang, Yue; Yan, Jingli; Li, Qi; Li, Jiafei; Gong, Shouzhe; Zhou, Hu; Gan, Jianhua; Jiang, Hualiang; Jia, Gui-Fang; Luo, Cheng; Yang, Cai-Guang

2015-01-01

Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N6-methyladenosine (m6A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m6A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m6A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine. PMID:25452335

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

Howard, A.D.

The aim of this research was to purify and characterize active opioid receptors and elucidate molecular aspects of opioid receptor heterogeneity. Purification to apparent homogeneity of an opioid binding protein from bovine caudate was achieved by solubilization in the non-ionic detergent, digitonin, followed by sequential chromatography on the opiate affinity matrix, ..beta..-naltrexylethylenediamine-CH-Sepharose 4B, and on the lectine affinity matrix, wheat germ agglutinin-agarose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by autoradiography revealed that radioiodinated purified receptor gave a single band. Purified receptor preparations showed a specific activity of 12,000-15,000 fmol of opiate bound per mgmore » of protein. Radioiodinated human beta-endorphin (/sup 125/I-beta-end/sub H/) was used as a probe to investigate the ligand binding subunits of mu and delta opioid receptors. /sup 125/I-beta-end/sub H/ was shown to bind to a variety of opioid receptor-containing tissues with high affinity and specificity with preference for mu and delta sites, and with little, if any, binding to kappa sites. Affinity crosslinking techniques were employed to covalently link /sup 125/I-beta-end/sub H/ to opioid receptors, utilizing derivatives of bis-succinimidyl esters that are bifunctional crosslinkers with specificities for amino and sulfhydryl groups. This, and competition experiments with high type-selective ligands, permitted the assignment of two labeled peptides to their receptor types, namely a peptide of M/sub r/ = 65,000 for mu receptors and one of M/sub r/ = 53,000 for delta receptors.« less

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

PubMed

Dixon-Clarke, Sarah E; Shehata, Saifeldin N; Krojer, Tobias; Sharpe, Timothy D; von Delft, Frank; Sakamoto, Kei; Bullock, Alex N

2017-02-20

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. © 2017 The Author(s).

Structural Elucidation and Molecular Docking of a Novel Antibiotic Compound from Cyanobacterium Nostoc sp. MGL001

PubMed Central

Niveshika; Verma, Ekta; Mishra, Arun K.; Singh, Angad K.; Singh, Vinay K.

2016-01-01

Cyanobacteria are rich source of array of bioactive compounds. The present study reports a novel antibacterial bioactive compound purified from cyanobacterium Nostoc sp. MGL001 using various chromatographic techniques viz. thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Further characterization was done using electrospray ionization mass spectroscopy (ESIMS) and nuclear magnetic resonance (NMR) and predicted structure of bioactive compound was 9-Ethyliminomethyl-12-(morpholin - 4 - ylmethoxy) -5, 8, 13, 16–tetraaza–hexacene - 2, 3 dicarboxylic acid (EMTAHDCA). Structure of EMTAHDCA clearly indicated that it is a novel compound that was not reported in literature or natural product database. The compound exhibited growth inhibiting effects mainly against the gram negative bacterial strains and produced maximum zone of inhibition at 150 μg/mL concentration. The compound was evaluated through in silico studies for its ability to bind 30S ribosomal fragment (PDB ID: 1YRJ, 1MWL, 1J7T, and 1LC4) and OmpF porin protein (4GCP, 4GCQ, and 4GCS) which are the common targets of various antibiotic drugs. Comparative molecular docking study revealed that EMTAHDCA has strong binding affinity for these selected targets in comparison to a number of most commonly used antibiotics. The ability of EMTAHDCA to bind the active sites on the proteins and 30S ribosomal fragments where the antibiotic drugs generally bind indicated that it is functionally similar to the commercially available drugs. PMID:27965634

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein.

PubMed

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E; Fernandez-Alberti, Sebastian

2015-06-28

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

NASA Astrophysics Data System (ADS)

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Roitberg, Adrian E.; Fernandez-Alberti, Sebastian

2015-06-01

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

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

DeVore, Natasha M.; Meneely, Kathleen M.; Bart, Aaron G.

2013-11-20

Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drugmore » pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 {angstrom}), CYP2A13 (3.0 {angstrom}), CYP2E1 (2.35 {angstrom}), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 {angstrom}) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.« less

Synthesis and biological evaluation of a series of aminoalkyl-tetralones and tetralols as dual dopamine/serotonin ligands.

PubMed

Carro, Laura; Torrado, María; Raviña, Enrique; Masaguer, Christian F; Lage, Sonia; Brea, José; Loza, María I

2014-01-01

A series of novel α-tetralone and α-tetralol derivatives was synthesized, and their binding affinities for 5-HT(2A) and D₂ receptors, the most important targets implicated in the anti-schizophrenia drug action, were evaluated to elucidate how substitutions in the aromatic ring of the pharmacophore affect to the affinity or selectivity for these receptors. The replacement of the H-7 in the tetrahydronaphthalene system by an amino group resulted in privileged 5-HT(2A) affinity of the 6-fluorobenzo[d]isoxazol derivative 36 and the alcohol 25 both showing a pK(i) value for 5-HT(2A) higher than 8.3 and good binding affinities for D₂ receptor leading to a Meltzer's ratio characteristic of an atypical antipsychotic profile. Additionally, a small collection of 3-aminomethyltetralone derivatives was prepared and examined here for their affinities and selectivities as 5-HT(2A)/D₂ dual ligands. Compound 11 shows the best profile with good pKi values for 5-HT(2A) and D₂ receptors leading to a Meltzer's ratio characteristic of a typical antipsychotic behaviour. These three compounds behaved as competitive antagonists of both 5-HT(2A) and D₂ receptors, and might be promising pharmacological tools for the investigation of the dual function of the 5HT(2A)-D₂ ligands. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide

NASA Astrophysics Data System (ADS)

Benjamin, Christopher J.; Wright, Kyle J.; Bolton, Scott C.; Hyun, Seok-Hee; Krynski, Kyle; Grover, Mahima; Yu, Guimei; Guo, Fei; Kinzer-Ursem, Tamara L.; Jiang, Wen; Thompson, David H.

2016-10-01

We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with Nα, Nα-dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His6-T7 bacteriophage and His6-GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His6-GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.

Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide.

PubMed

Benjamin, Christopher J; Wright, Kyle J; Bolton, Scott C; Hyun, Seok-Hee; Krynski, Kyle; Grover, Mahima; Yu, Guimei; Guo, Fei; Kinzer-Ursem, Tamara L; Jiang, Wen; Thompson, David H

2016-10-17

We report the fabrication of transmission electron microscopy (TEM) grids bearing graphene oxide (GO) sheets that have been modified with N α , N α -dicarboxymethyllysine (NTA) and deactivating agents to block non-selective binding between GO-NTA sheets and non-target proteins. The resulting GO-NTA-coated grids with these improved antifouling properties were then used to isolate His 6 -T7 bacteriophage and His 6 -GroEL directly from cell lysates. To demonstrate the utility and simplified workflow enabled by these grids, we performed cryo-electron microscopy (cryo-EM) of His 6 -GroEL obtained from clarified E. coli lysates. Single particle analysis produced a 3D map with a gold standard resolution of 8.1 Å. We infer from these findings that TEM grids modified with GO-NTA are a useful tool that reduces background and improves both the speed and simplicity of biological sample preparation for high-resolution structure elucidation by cryo-EM.

Molecular Bases of PDE4D Inhibition by Memory-Enhancing GEBR Library Compounds.

PubMed

Prosdocimi, Tommaso; Mollica, Luca; Donini, Stefano; Semrau, Marta S; Lucarelli, Anna Paola; Aiolfi, Egidio; Cavalli, Andrea; Storici, Paola; Alfei, Silvana; Brullo, Chiara; Bruno, Olga; Parisini, Emilio

2018-05-01

Selected members of the large rolipram-related GEBR family of type 4 phosphodiesterase (PDE4) inhibitors have been shown to facilitate long-term potentiation and to improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, few if any structure-activity relationship studies have been performed to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degree of selectivity between the different PDE4 isoforms.

Dual Targeting of the Chemokine Receptors CXCR4 and ACKR3 with Novel Engineered Chemokines*

PubMed Central

Hanes, Melinda S.; Salanga, Catherina L.; Chowdry, Arnab B.; Comerford, Iain; McColl, Shaun R.; Kufareva, Irina; Handel, Tracy M.

2015-01-01

The chemokine CXCL12 and its G protein-coupled receptors CXCR4 and ACKR3 are implicated in cancer and inflammatory and autoimmune disorders and are targets of numerous antagonist discovery efforts. Here, we describe a series of novel, high affinity CXCL12-based modulators of CXCR4 and ACKR3 generated by selection of N-terminal CXCL12 phage libraries on live cells expressing the receptors. Twelve of 13 characterized CXCL12 variants are full CXCR4 antagonists, and four have Kd values <5 nm. The new variants also showed high affinity for ACKR3. The variant with the highest affinity for CXCR4, LGGG-CXCL12, showed efficacy in a murine model for multiple sclerosis, demonstrating translational potential. Molecular modeling was used to elucidate the structural basis of binding and antagonism of selected variants and to guide future designs. Together, this work represents an important step toward the development of therapeutics targeting CXCR4 and ACKR3. PMID:26216880

Scaffold-Focused Virtual Screening: Prospective Application to the Discovery of TTK Inhibitors

PubMed Central

2013-01-01

We describe and apply a scaffold-focused virtual screen based upon scaffold trees to the mitotic kinase TTK (MPS1). Using level 1 of the scaffold tree, we perform both 2D and 3D similarity searches between a query scaffold and a level 1 scaffold library derived from a 2 million compound library; 98 compounds from 27 unique top-ranked level 1 scaffolds are selected for biochemical screening. We show that this scaffold-focused virtual screen prospectively identifies eight confirmed active compounds that are structurally differentiated from the query compound. In comparison, 100 compounds were selected for biochemical screening using a virtual screen based upon whole molecule similarity resulting in 12 confirmed active compounds that are structurally similar to the query compound. We elucidated the binding mode for four of the eight confirmed scaffold hops to TTK by determining their protein–ligand crystal structures; each represents a ligand-efficient scaffold for inhibitor design. PMID:23672464

Exploring the structure determinants of pyrazinone derivatives as PDE5 3HC8 inhibitors: an in silico analysis.

PubMed

Li, Yan; Wu, Wenzhao; Ren, Hong; Wang, Jinghui; Zhang, Shuwei; Li, Guohui; Yang, Ling

2012-09-01

Phosphodiesterase type 5 (PDE5) inhibitors are clinically indicated for the treatment of erectile dysfunction, pulmonary hypertension and various other diseases. In this work, both ligand- and receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques on 122 pyrazinone derivatives as PDE inhibitors. The resultant optimum 3D-QSAR model exhibits a proper predictive ability as indicated by the statistical results of Q² of 0.584, R(ncv)² of 0.884 and R(pre)² of 0.817, respectively. In addition, docking analysis and molecular dynamics (MD) simulation were also applied to elucidate the probable binding modes of these inhibitors. Our main findings are: (1) Introduction of bulky, electropositive and hydrophobic substituents at 12- and 19-positions can increase the biological activities. (2) N atom at 8-position is detrimental to the inhibitor activity, and the effect of N atoms at 5- and 6-positions on compound activity is co-determined by both the hydrophobic force and the π-π stacking interaction. (3) Bulky and hydrophilic substitutions are favored at the 27-position of ring D. (4) Electronegative and hydrophilic substitutions around 5- and 6-positions increase the inhibitory activity. (5) Hydrophobic forces and π-π stacking interaction with Phe786 and Phe820 are crucial in determining the binding of pyrazinone derivatives to PDE5. (6) Bulky substitutions around ring C favors selectivity against PDE11, while bulky groups near the 21-position disfavor the selectivity. The information obtained from this work can be utilized to accurately predict the binding affinity of related analogues and also facilitate future rational designs of novel PDE5 inhibitors with improved activity and selectivity. Copyright © 2012 Elsevier Inc. All rights reserved.

Ligand-Dependent Activation and Deactivation of the Human Adenosine A2A Receptor

PubMed Central

Li, Jianing; Jonsson, Amanda L.; Beuming, Thijs; Shelley, John C.; Voth, Gregory A.

2013-01-01

G protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR’s activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp2466.48). Our findings agree with a previously proposed view, that during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G protein-binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal structures. These results should aid in the discovery of more effective and selective GPCR ligands. PMID:23678995

Ligand-dependent activation and deactivation of the human adenosine A(2A) receptor.

PubMed

Li, Jianing; Jonsson, Amanda L; Beuming, Thijs; Shelley, John C; Voth, Gregory A

2013-06-12

G-protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR's activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular, we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp246(6.48)). Our findings agree with a previously proposed view that, during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G-protein binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity, and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal structures. These results should aid in the discovery of more effective and selective GPCR ligands.

Native Mass Spectrometry in Fragment-Based Drug Discovery.

PubMed

Pedro, Liliana; Quinn, Ronald J

2016-07-28

The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Calculating binding free energies for protein-carbohydrate complexes.

PubMed

Hadden, Jodi A; Tessier, Matthew B; Fadda, Elisa; Woods, Robert J

2015-01-01

A variety of computational techniques may be applied to compute theoretical binding free energies for protein-carbohydrate complexes. Elucidation of the intermolecular interactions, as well as the thermodynamic effects, that contribute to the relative strength of receptor binding can shed light on biomolecular recognition, and the resulting initiation or inhibition of a biological process. Three types of free energy methods are discussed here, including MM-PB/GBSA, thermodynamic integration, and a non-equilibrium alternative utilizing SMD. Throughout this chapter, the well-known concanavalin A lectin is employed as a model system to demonstrate the application of these methods to the special case of carbohydrate binding.

Structural Insights into the Phospholipid Binding Specificity of Human Evectin-2

NASA Astrophysics Data System (ADS)

Okazaki, Seiji; Kato, Ryuichi; Wakatsuki, Soichi; Uchida, Yasunori; Taguchi, Tomohiko; Arai, Hiroyuki

Evectin-2 is a recycling endosomal protein and plays an essential role in retrograde transport from recycling endosomes to the trans-Golgi network. The pleckstrin homology (PH) domain of Evectin-2 can specifically binds to phosphatidylserine (PS), which is enriched in recycling endosomes. To elucidate the molecular mechanism how it specifically binds to PS, we solved the crystal structures of human Evectin-2 PH domain for apo and O-phospho-L-serine complexed forms at 1.75 and 1.00 Å resolution, respectively. These structural analyses clearly show that PS-induced conformational change of Evectin-2 PH domain effectively explains the strict phospholipid binding specificity.

Annotation and structural elucidation of bovine milk oligosaccharides and determination of novel fucosylated structures

PubMed Central

Aldredge, Danielle L; Geronimo, Maria R; Hua, Serenus; Nwosu, Charles C; Lebrilla, Carlito B; Barile, Daniela

2013-01-01

Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high-potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of biotherapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structure-related specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomer-specific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass and structure to allow their rapid identification in future studies. PMID:23436288

A compound-based proteomic approach discloses 15-ketoatractyligenin methyl ester as a new PPARγ partial agonist with anti-proliferative ability

NASA Astrophysics Data System (ADS)

Vasaturo, Michele; Fiengo, Lorenzo; de Tommasi, Nunziatina; Sabatino, Lina; Ziccardi, Pamela; Colantuoni, Vittorio; Bruno, Maurizio; Cerchia, Carmen; Novellino, Ettore; Lupo, Angelo; Lavecchia, Antonio; Piaz, Fabrizio Dal

2017-01-01

Proteomics based approaches are emerging as useful tools to identify the targets of bioactive compounds and elucidate their molecular mechanisms of action. Here, we applied a chemical proteomic strategy to identify the peroxisome proliferator-activated receptor γ (PPARγ) as a molecular target of the pro-apoptotic agent 15-ketoatractyligenin methyl ester (compound 1). We demonstrated that compound 1 interacts with PPARγ, forms a covalent bond with the thiol group of C285 and occupies the sub-pocket between helix H3 and the β-sheet of the ligand-binding domain (LBD) of the receptor by Surface Plasmon Resonance (SPR), mass spectrometry-based studies and docking experiments. 1 displayed partial agonism of PPARγ in cell-based transactivation assays and was found to inhibit the AKT pathway, as well as its downstream targets. Consistently, a selective PPARγ antagonist (GW9662) greatly reduced the anti-proliferative and pro-apoptotic effects of 1, providing the molecular basis of its action. Collectively, we identified 1 as a novel PPARγ partial agonist and elucidated its mode of action, paving the way for therapeutic strategies aimed at tailoring novel PPARγ ligands with reduced undesired harmful side effects.

Orphan nuclear receptor ERRγ is a key regulator of human fibrinogen gene expression

PubMed Central

Zhang, Yaochen; Kim, Don-Kyu; Lu, Yan; Jung, Yoon Seok; Lee, Ji-min; Kim, Young-Hoon; Lee, Yong Soo; Kim, Jina; Dewidar, Bedair; Jeong, Won-IL; Lee, In-Kyu; Cho, Sung Jin; Dooley, Steven; Lee, Chul-Ho; Li, Xiaoying

2017-01-01

Fibrinogen, 1 of 13 coagulation factors responsible for normal blood clotting, is synthesized by hepatocytes. Detailed roles of the orphan nuclear receptors regulating fibrinogen gene expression have not yet been fully elucidated. Here, we identified estrogen-related receptor gamma (ERRγ) as a novel transcriptional regulator of human fibrinogen gene expression. Overexpression of ERRγ specially increased fibrinogen expression in human hepatoma cell line. Cannabinoid receptor types 1(CB1R) agonist arachidonyl-2'-chloroethylamide (ACEA) up-regulated transcription of fibrinogen via induction of ERRγ, whereas knockdown of ERRγ attenuated fibrinogen expression. Deletion analyses of the fibrinogen γ (FGG) gene promoter and ChIP assays revealed binding sites of ERRγ on human fibrinogen γ gene promoter. Moreover, overexpression of ERRγ was sufficient to increase fibrinogen gene expression, whereas treatment with GSK5182, a selective inverse agonist of ERRγ led to its attenuation in cell culture. Finally, fibrinogen and ERRγ gene expression were elevated in liver tissue of obese patients suggesting a conservation of this mechanism. Overall, this study elucidates a molecular mechanism linking CB1R signaling, ERRγ expression and fibrinogen gene transcription. GSK5182 may have therapeutic potential to treat hyperfibrinogenemia. PMID:28750085

A compound-based proteomic approach discloses 15-ketoatractyligenin methyl ester as a new PPARγ partial agonist with anti-proliferative ability

PubMed Central

Vasaturo, Michele; Fiengo, Lorenzo; De Tommasi, Nunziatina; Sabatino, Lina; Ziccardi, Pamela; Colantuoni, Vittorio; Bruno, Maurizio; Cerchia, Carmen; Novellino, Ettore; Lupo, Angelo; Lavecchia, Antonio; Piaz, Fabrizio Dal

2017-01-01

Proteomics based approaches are emerging as useful tools to identify the targets of bioactive compounds and elucidate their molecular mechanisms of action. Here, we applied a chemical proteomic strategy to identify the peroxisome proliferator-activated receptor γ (PPARγ) as a molecular target of the pro-apoptotic agent 15-ketoatractyligenin methyl ester (compound 1). We demonstrated that compound 1 interacts with PPARγ, forms a covalent bond with the thiol group of C285 and occupies the sub-pocket between helix H3 and the β-sheet of the ligand-binding domain (LBD) of the receptor by Surface Plasmon Resonance (SPR), mass spectrometry-based studies and docking experiments. 1 displayed partial agonism of PPARγ in cell-based transactivation assays and was found to inhibit the AKT pathway, as well as its downstream targets. Consistently, a selective PPARγ antagonist (GW9662) greatly reduced the anti-proliferative and pro-apoptotic effects of 1, providing the molecular basis of its action. Collectively, we identified 1 as a novel PPARγ partial agonist and elucidated its mode of action, paving the way for therapeutic strategies aimed at tailoring novel PPARγ ligands with reduced undesired harmful side effects. PMID:28117438

A compound-based proteomic approach discloses 15-ketoatractyligenin methyl ester as a new PPARγ partial agonist with anti-proliferative ability.

PubMed

Vasaturo, Michele; Fiengo, Lorenzo; De Tommasi, Nunziatina; Sabatino, Lina; Ziccardi, Pamela; Colantuoni, Vittorio; Bruno, Maurizio; Cerchia, Carmen; Novellino, Ettore; Lupo, Angelo; Lavecchia, Antonio; Piaz, Fabrizio Dal

2017-01-24

Proteomics based approaches are emerging as useful tools to identify the targets of bioactive compounds and elucidate their molecular mechanisms of action. Here, we applied a chemical proteomic strategy to identify the peroxisome proliferator-activated receptor γ (PPARγ) as a molecular target of the pro-apoptotic agent 15-ketoatractyligenin methyl ester (compound 1). We demonstrated that compound 1 interacts with PPARγ, forms a covalent bond with the thiol group of C285 and occupies the sub-pocket between helix H3 and the β-sheet of the ligand-binding domain (LBD) of the receptor by Surface Plasmon Resonance (SPR), mass spectrometry-based studies and docking experiments. 1 displayed partial agonism of PPARγ in cell-based transactivation assays and was found to inhibit the AKT pathway, as well as its downstream targets. Consistently, a selective PPARγ antagonist (GW9662) greatly reduced the anti-proliferative and pro-apoptotic effects of 1, providing the molecular basis of its action. Collectively, we identified 1 as a novel PPARγ partial agonist and elucidated its mode of action, paving the way for therapeutic strategies aimed at tailoring novel PPARγ ligands with reduced undesired harmful side effects.

Pyrazole-based cathepsin S inhibitors with arylalkynes as P1 binding elements

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

Ameriks, Michael K.; Axe, Frank U.; Bembenek, Scott D.

A crystal structure of 1 bound to a Cys25Ser mutant of cathepsin S helped to elucidate the binding mode of a previously disclosed series of pyrazole-based CatS inhibitors and facilitated the design of a new class of arylalkyne analogs. Optimization of the alkyne and tetrahydropyridine portions of the pharmacophore provided potent CatS inhibitors (IC{sub 50} = 40-300 nM), and an X-ray structure of 32 revealed that the arylalkyne moiety binds in the S1 pocket of the enzyme.

Redesigned Spider Peptide with Improved Antimicrobial and Anticancer Properties.

PubMed

Troeira Henriques, Sónia; Lawrence, Nicole; Chaousis, Stephanie; Ravipati, Anjaneya S; Cheneval, Olivier; Benfield, Aurélie H; Elliott, Alysha G; Kavanagh, Angela Maria; Cooper, Matthew A; Chan, Lai Yue; Huang, Yen-Hua; Craik, David J

2017-09-15

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.

IFN-λ cancer immunotherapy: new kid on the block.

PubMed

Lasfar, Ahmed; Gogas, Helen; Zloza, Andrew; Kaufman, Howard L; Kirkwood, John M

2016-07-01

Interferon-lambda (IFN-λ) is a new IFN type, related to IFN-α, that is commonly used in the clinic. However, significant side effects accompanying IFN-α treatment limit enthusiasm for IFN-α. In this review, we discuss the current landscape of IFN-α use in oncology and describe the biologic characteristics of IFN-λ. IFN-λ offers unique advantages, including a more tumor cell selective targeting, lower off-target binding and an ability to generate both innate and adaptive immune responses. IFN-λ has also demonstrated therapeutic benefit in murine cancer models. IFN-λ may be used in clinic as a single agent or in combination with other immunotherapy agents, such as immune checkpoint inhibitors. Further clinical trials will be needed to fully elucidate the potential of this novel agent in oncology.

Ca2+-binding Motif of βγ-Crystallins*

PubMed Central

Srivastava, Shanti Swaroop; Mishra, Amita; Krishnan, Bal; Sharma, Yogendra

2014-01-01

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca2+ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca2+-binding sites. βγ-Crystallins make a separate class of Ca2+-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca2+ binding to βγ-crystallins in mediating biological processes are yet to be elucidated. PMID:24567326

Crystal structures of sampatrilat and sampatrilat-Asp in complex with human ACE - a molecular basis for domain selectivity.

PubMed

Cozier, Gyles E; Schwager, Sylva L; Sharma, Rajni K; Chibale, Kelly; Sturrock, Edward D; Acharya, K Ravi

2018-04-01

Angiotensin-1-converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (known as nACE and cACE) with different substrate specificities. Based on kinetic studies it was previously reported that sampatrilat, a tight-binding inhibitor of ACE, K i = 13.8 nm and 171.9 nm for cACE and nACE respectively [Sharma et al., Journal of Chemical Information and Modeling (2016), 56, 2486-2494], was 12.4-fold more selective for cACE. In addition, samAsp, in which an aspartate group replaces the sampatrilat lysine, was found to be a nonspecific and lower micromolar affinity inhibitor. Here, we report a detailed three-dimensional structural analysis of sampatrilat and samAsp binding to ACE using high-resolution crystal structures elucidated by X-ray crystallography, which provides a molecular basis for differences in inhibitor affinity and selectivity for nACE and cACE. The structures show that the specificity of sampatrilat can be explained by increased hydrophobic interactions and a H-bond from Glu403 of cACE with the lysine side chain of sampatrilat that are not observed in nACE. In addition, the structures clearly show a significantly greater number of hydrophilic and hydrophobic interactions with sampatrilat compared to samAsp in both cACE and nACE consistent with the difference in affinities. Our findings provide new experimental insights into ligand binding at the active site pockets that are important for the design of highly specific domain selective inhibitors of ACE. The atomic coordinates and structure factors for N- and C-domains of ACE bound to sampatrilat and sampatrilat-Asp complexes (6F9V, 6F9R, 6F9T and 6F9U respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). © 2018 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Insights into the binding of GABA to the insect RDL receptor from atomistic simulations: a comparison of models

NASA Astrophysics Data System (ADS)

Comitani, Federico; Cohen, Netta; Ashby, Jamie; Botten, Dominic; Lummis, Sarah C. R.; Molteni, Carla

2014-01-01

The resistance to dieldrin (RDL) receptor is an insect pentameric ligand-gated ion channel (pLGIC). It is activated by the neurotransmitter γ-aminobutyric acid (GABA) binding to its extracellular domain; hence elucidating the atomistic details of this interaction is important for understanding how the RDL receptor functions. As no high resolution structures are currently available, we built homology models of the extracellular domain of the RDL receptor using different templates, including the widely used acetylcholine binding protein and two pLGICs, the Erwinia Chrysanthemi ligand-gated ion channel (ELIC) and the more recently resolved GluCl. We then docked GABA into the selected three dimensional structures, which we used as starting points for classical molecular dynamics simulations. This allowed us to analyze in detail the behavior of GABA in the binding sites, including the hydrogen bond and cation-π interaction networks it formed, the conformers it visited and the possible role of water molecules in mediating the interactions; we also estimated the binding free energies. The models were all stable and showed common features, including interactions consistent with experimental data and similar to other pLGICs; differences could be attributed to the quality of the models, which increases with increasing sequence identity, and the use of a pLGIC template. We supplemented the molecular dynamics information with metadynamics, a rare event method, by exploring the free energy landscape of GABA binding to the RDL receptor. Overall, we show that the GluCl template provided the best models. GABA forming direct salt-bridges with Arg211 and Glu204, and cation-π interactions with an aromatic cage including Tyr109, Phe206 and Tyr254, represents a favorable binding arrangement, and the interaction with Glu204 can also be mediated by a water molecule.

The SRL peptide of Rhesus Rotavirus VP4 protein governs cholangiocyte infection and the murine model of biliary atresia

PubMed Central

Mohanty, Sujit K.; Donnelly, Bryan; Lobeck, Inna; Walther, Ashley; Dupree, Phylicia; Coots, Abigail; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

2016-01-01

Biliary atresia (BA) is a neonatal obstructive cholangiopathy which progresses to end stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Utilizing rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains which cause an obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4 derived peptide (TRTRVSRLY), significantly reduced RRV’s ability to bind and infect the cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains which do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by siRNAs reduced RRV’s ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice. Conclusion The tri-peptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70 defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response following this interaction to understand how it contributes to the pathogenesis of BA. PMID:27859498

PDZ binding to the BAR domain of PICK1 is elucidated by coarse-grained molecular dynamics.

PubMed

He, Yi; Liwo, Adam; Weinstein, Harel; Scheraga, Harold A

2011-01-07

A key regulator of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor traffic, PICK1 is known to interact with over 40 other proteins, including receptors, transporters and ionic channels, and to be active mostly as a homodimer. The current lack of a complete PICK1 structure determined at atomic resolution hinders the elucidation of its functional mechanisms. Here, we identify interactions between the component PDZ and BAR domains of PICK1 by calculating possible binding sites for the PDZ domain of PICK1 (PICK1-PDZ) to the homology-modeled, crescent-shaped dimer of the PICK1-BAR domain using multiplexed replica-exchange molecular dynamics (MREMD) and canonical molecular dynamics simulations with the coarse-grained UNRES force field. The MREMD results show that the preferred binding site for the single PDZ domain is the concave cavity of the BAR dimer. A second possible binding site is near the N-terminus of the BAR domain that is linked directly to the PDZ domain. Subsequent short canonical molecular dynamics simulations used to determine how the PICK1-PDZ domain moves to the preferred binding site on the BAR domain of PICK1 revealed that initial hydrophobic interactions drive the progress of the simulated binding. Thus, the concave face of the BAR dimer accommodates the PDZ domain first by weak hydrophobic interactions and then the PDZ domain slides to the center of the concave face, where more favorable hydrophobic interactions take over. Copyright © 2010 Elsevier Ltd. All rights reserved.

Multiple Binding Modes between HNF4[alpha] and the LXXLL Motifs of PGC-1[alpha] Lead to Full Activation

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

Rha, Geun Bae; Wu, Guangteng; Shoelson, Steven E.

2010-04-15

Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4{alpha} interacts, peroxisome proliferation-activated receptor {gamma} (PPAR{gamma}) coactivator 1{alpha} (PGC-1{alpha}) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1{alpha} recruitment, we have determined the crystal structure of HNF4{alpha} in complex with amore » fragment of PGC-1{alpha} containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4{alpha} toward the LXXLL motifs of PGC-1{alpha} could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.« less

Plasticity in interactions of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlies promiscuity of FGF1.

PubMed

Beenken, Andrew; Eliseenkova, Anna V; Ibrahimi, Omar A; Olsen, Shaun K; Mohammadi, Moosa

2012-01-27

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of fibroblast growth factor receptors 1-3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived fibroblast growth factors (FGFs), respectively. FGF1 is termed the "universal FGFR ligand" because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the "b" and "c" splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.

A rational approach to elucidate human monoamine oxidase molecular selectivity.

PubMed

Mangiatordi, Giuseppe Felice; Alberga, Domenico; Pisani, Leonardo; Gadaleta, Domenico; Trisciuzzi, Daniela; Farina, Roberta; Carotti, Andrea; Lattanzi, Gianluca; Catto, Marco; Nicolotti, Orazio

2017-04-01

Designing highly selective human monoamine oxidase (hMAO) inhibitors is a challenging goal on the road to a more effective treatment of depression and anxiety (inhibition of hMAO-A isoform) as well as neurodegenerative diseases (inhibition of hMAO-B isoform). To uncover the molecular rationale of hMAOs selectivity, two recently prepared 2H-chromene-2-ones, namely compounds 1 and 2, were herein chosen as molecular probes being highly selective toward hMAO-A and hMAO-B, respectively. We performed molecular dynamics (MD) studies on four different complexes, cross-simulating one at a time the two hMAO-isoforms (dimer embedded in a lipid bilayer) with the two considered probes. Our comparative analysis on the obtained 100ns trajectories discloses a stable H-bond interaction between 1 and Gln215 as crucial for ligand selectivity toward hMAO-A whereas a water-mediated interaction might explain the observed hMAO-B selectivity of compound 2. Such hypotheses are further supported by binding free energy calculations carried out applying the molecular mechanics generalized Born surface area (MM-GBSA) method and allowing us to evaluate the contribution of each residue to the observed isoform selectivity. Taken as whole, this study represents the first attempt to explain at molecular level hMAO isoform selectivity and a valuable yardstick for better addressing the design of new and highly selective MAO inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

More than a simple lipophilic contact: a detailed thermodynamic analysis of nonbasic residues in the s1 pocket of thrombin.

PubMed

Baum, Bernhard; Mohamed, Menshawy; Zayed, Mohamed; Gerlach, Christof; Heine, Andreas; Hangauer, David; Klebe, Gerhard

2009-07-03

The field of medicinal chemistry aims to design and optimize small molecule leads into drug candidates that may positively interfere with pathological disease situations in humans or combat the growth of infective pathogens. From the plethora of crystal structures of protein-inhibitor complexes we have learned how molecules recognize each other geometrically, but we still have rather superficial understanding of why they bind to each other. This contribution surveys a series of 26 thrombin inhibitors with small systematic structural differences to elucidate the rationale for their widely deviating binding affinity from 185 microM to 4 nM as recorded by enzyme kinetic measurements. Five well-resolved (resolution 2.30 - 1.47 A) crystal structures of thrombin-inhibitor complexes and an apo-structure of the uncomplexed enzyme (1.50 A) are correlated with thermodynamic data recorded by isothermal titration calorimetry with 12 selected inhibitors from the series. Taking solubility data into account, the variation in physicochemical properties allows conclusions to be reached about the relative importance of the enthalpic binding features as well as to estimate the importance of the parameters more difficult to capture, such as residual ligand entropy and desolvation properties. The collected data reveal a comprehensive picture of the thermodynamic signature that explains the so far poorly understood attractive force experienced by m-chloro-benzylamides to thrombin.

Structural analysis of poly-SUMO chain recognition by the RNF4-SIMs domain.

PubMed

Kung, Camy C-H; Naik, Mandar T; Wang, Szu-Huan; Shih, Hsiu-Ming; Chang, Che-Chang; Lin, Li-Ying; Chen, Chia-Lin; Ma, Che; Chang, Chi-Fon; Huang, Tai-Huang

2014-08-15

The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the β2-strand and the α1-helix parallel to the β2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indicate that it exists as an ordered structure. The HADDOCK model showed that the tandem RNF4-SIMs domain bound antiparallel to the tetra-SUMO2 chain orientation and wrapped around the SUMO protamers in a superhelical turn without imposing steric hindrance on either molecule.

Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels

PubMed Central

von Stein, Richard T.; Silver, Kristopher S.; Soderlund, David M.

2013-01-01

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents. PMID:24072940

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

Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo

The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use inmore » combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.« less

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

PubMed

Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

2016-01-01

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

Critical ligand binding reagent preparation/selection: when specificity depends on reagents.

PubMed

Rup, Bonita; O'Hara, Denise

2007-05-11

Throughout the life cycle of biopharmaceutical products, bioanalytical support is provided using ligand binding assays to measure the drug product for pharmacokinetic, pharmacodynamic, and immunogenicity studies. The specificity and selectivity of these ligand binding assays are highly dependent on the ligand binding reagents. Thus the selection, characterization, and management processes for ligand binding reagents are crucial to successful assay development and application. This report describes process considerations for selection and characterization of ligand binding reagents that are integral parts of the different phases of assay development. Changes in expression, purification, modification, and storage of the ligand binding reagents may have a profound effect on the ligand binding assay performance. Thus long-term management of the critical ligand binding assay reagents is addressed including suggested characterization criteria that allow ligand binding reagents to be used in as consistent a manner as possible. Examples of challenges related to the selection, modification, and characterization of ligand binding reagents are included.

Echinococcus granulosus fatty acid binding proteins subcellular localization.

PubMed

Alvite, Gabriela; Esteves, Adriana

2016-05-01

Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

Free energy landscape of siRNA-polycation complexation: Elucidating the effect of molecular geometry, polymer flexibility, and charge neutralization.

PubMed

Grasso, Gianvito; Deriu, Marco Agostino; Patrulea, Viorica; Borchard, Gerrit; Möller, Michael; Danani, Andrea

2017-01-01

The success of medical threatments with DNA and silencing interference RNA is strongly related to the design of efficient delivery technologies. Cationic polymers represent an attractive strategy to serve as nucleic-acid carriers with the envisioned advantages of efficient complexation, low cost, ease of production, well-defined size, and low polydispersity index. However, the balance between efficacy and toxicity (safety) of these polymers is a challenge and in need of improvement. With the aim of designing more effective polycationic-based gene carriers, many parameters such as carrier morphology, size, molecular weight, surface chemistry, and flexibility/rigidity ratio need to be taken into consideration. In the present work, the binding mechanism of three cationic polymers (polyarginine, polylysine and polyethyleneimine) to a model siRNA target is computationally investigated at the atomistic level. In order to better understand the polycationic carrier-siRNA interactions, replica exchange molecular dynamic simulations were carried out to provide an exhaustive exploration of all the possible binding sites, taking fully into account the siRNA flexibility together with the presence of explicit solvent and ions. Moreover, well-tempered metadynamics simulations were employed to elucidate how molecular geometry, polycation flexibility, and charge neutralization affect the siRNA-polycations free energy landscape in term of low-energy binding modes and unbinding free energy barriers. Significant differences among polymer binding modes have been detected, revealing the advantageous binding properties of polyarginine and polylysine compared to polyethyleneimine.

Free energy landscape of siRNA-polycation complexation: Elucidating the effect of molecular geometry, polymer flexibility, and charge neutralization

PubMed Central

Patrulea, Viorica; Borchard, Gerrit; Möller, Michael; Danani, Andrea

2017-01-01

The success of medical threatments with DNA and silencing interference RNA is strongly related to the design of efficient delivery technologies. Cationic polymers represent an attractive strategy to serve as nucleic-acid carriers with the envisioned advantages of efficient complexation, low cost, ease of production, well-defined size, and low polydispersity index. However, the balance between efficacy and toxicity (safety) of these polymers is a challenge and in need of improvement. With the aim of designing more effective polycationic-based gene carriers, many parameters such as carrier morphology, size, molecular weight, surface chemistry, and flexibility/rigidity ratio need to be taken into consideration. In the present work, the binding mechanism of three cationic polymers (polyarginine, polylysine and polyethyleneimine) to a model siRNA target is computationally investigated at the atomistic level. In order to better understand the polycationic carrier-siRNA interactions, replica exchange molecular dynamic simulations were carried out to provide an exhaustive exploration of all the possible binding sites, taking fully into account the siRNA flexibility together with the presence of explicit solvent and ions. Moreover, well-tempered metadynamics simulations were employed to elucidate how molecular geometry, polycation flexibility, and charge neutralization affect the siRNA-polycations free energy landscape in term of low-energy binding modes and unbinding free energy barriers. Significant differences among polymer binding modes have been detected, revealing the advantageous binding properties of polyarginine and polylysine compared to polyethyleneimine. PMID:29088239

Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie)▿

PubMed Central

Anilkumar, Konasale J.; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J.

2008-01-01

Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments. PMID:18024681

Network Analysis Reveals the Recognition Mechanism for Mannose-binding Lectins

NASA Astrophysics Data System (ADS)

Zhao, Yunjie; Jian, Yiren; Zeng, Chen; Computational Biophysics Lab Team

The specific carbohydrate binding of mannose-binding lectin (MBL) protein in plants makes it a very useful molecular tool for cancer cell detection and other applications. The biological states of most MBL proteins are dimeric. Using dynamics network analysis on molecular dynamics (MD) simulations on the model protein of MBL, we elucidate the short- and long-range driving forces behind the dimer formation. The results are further supported by sequence coevolution analysis. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.

Limited tryptic proteolysis of the benzodiazepine binding proteins in different species reveals structural homologies.

PubMed

Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J

1988-12-01

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.

Structure, recognition and adaptive binding in RNA aptamer complexes.

PubMed

Patel, D J; Suri, A K; Jiang, F; Jiang, L; Fan, P; Kumar, R A; Nonin, S

1997-10-10

Novel features of RNA structure, recognition and discrimination have been recently elucidated through the solution structural characterization of RNA aptamers that bind cofactors, aminoglycoside antibiotics, amino acids and peptides with high affinity and specificity. This review presents the solution structures of RNA aptamer complexes with adenosine monophosphate, flavin mononucleotide, arginine/citrulline and tobramycin together with an example of hydrogen exchange measurements of the base-pair kinetics for the AMP-RNA aptamer complex. A comparative analysis of the structures of these RNA aptamer complexes yields the principles, patterns and diversity associated with RNA architecture, molecular recognition and adaptive binding associated with complex formation.

Structural and Functional Evidence for Testosterone Activation of GPRC6A in Peripheral Tissues

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

Pi, Min; Kapoor, Karan; Wu, Yunpeng

G protein-coupled receptor (GPCR) family C group 6 member A (GPRC6A) is a multiligand GPCR that is activated by cations, L-amino acids, and osteocalcin. GPRC6A plays an important role in the regulation of testosterone (T) production and energy metabolism in mice. T has rapid, transcription-independent (nongenomic) effects that are mediated by a putative GPCR. We previously found that T can activate GPRC6A in vitro, but the possibility that T is a ligand for GPRC6A remains controversial. Here, we demonstrate direct T binding to GPRC6A and construct computational structural models of GPRC6A that are used to identify potential binding poses ofmore » T. Mutations of the predicted binding site residues were experimentally found to block T activation of GPRC6A, in agreement with the modeling. Using Gpr6ca(-/-) mice, we confirmed that loss of GPRC6A resulted in loss of T rapid signaling responses and elucidated several biological functions regulated by GPRC6A-dependent T rapid signaling, including T stimulation of insulin secretion in pancreatic islets and enzyme expression involved in the biosynthesis of T in Leydig cells. Finally, we identified a stereo-specific effect of an R-isomer of a selective androgen receptor modulator that is predicted to bind to and shown to activate GPRC6A but not androgen receptor. Together, our data show that GPRC6A directly mediates the rapid signaling response to T and uncovers previously unrecognized endocrine networks.« less

Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level

PubMed Central

Schvartzman, Mark; Palma, Matteo; Sable, Julia; Abramson, Justin; Hu, Xian; Sheetz, Michael P.; Wind, Shalom J.

2011-01-01

The ability to control the placement of individual molecules promises to enable a wide range of applications and is a key challenge in nanoscience and nanotechnology. Many biological interactions, in particular, are sensitive to the precise geometric arrangement of proteins. We have developed a technique which combines molecular-scale nanolithography with site-selective biochemistry to create biomimetic arrays of individual protein binding sites. The binding sites can be arranged in heterogeneous patterns of virtually any possible geometry with a nearly unlimited number of degrees of freedom. We have used these arrays to explore how the geometric organization of the extracellular matrix (ECM) binding ligand RGD (Arg-Gly-Asp) affects cell adhesion and spreading. Systematic variation of spacing, density and cluster size of individual integrin binding sites was used to elicit different cell behavior. Cell spreading assays on arrays of different geometric arrangements revealed a dramatic increase in spreading efficiency when at least 4 liganded sites were spaced within 60 nm or less, with no dependence on global density. This points to the existence of a minimal matrix adhesion unit for fibronectin defined in space and stoichiometry. Developing an understanding of the ECM geometries that activate specific cellular functional complexes is a critical step toward controlling cell behavior. Potential practical applications range from new therapeutic treatments to the rational design of tissue scaffolds that can optimize healing without scarring. More broadly, spatial control at the single-molecule level can elucidate factors controlling individual molecular interactions and can enable synthesis of new systems based on molecular-scale architectures. PMID:21319842

Structural and Functional Evidence for Testosterone Activation of GPRC6A in Peripheral Tissues

DOE PAGES

Pi, Min; Kapoor, Karan; Wu, Yunpeng; ...

2015-01-01

G protein-coupled receptor (GPCR) family C group 6 member A (GPRC6A) is a multiligand GPCR that is activated by cations, L-amino acids, and osteocalcin. GPRC6A plays an important role in the regulation of testosterone (T) production and energy metabolism in mice. T has rapid, transcription-independent (nongenomic) effects that are mediated by a putative GPCR. We previously found that T can activate GPRC6A in vitro, but the possibility that T is a ligand for GPRC6A remains controversial. Here, we demonstrate direct T binding to GPRC6A and construct computational structural models of GPRC6A that are used to identify potential binding poses ofmore » T. Mutations of the predicted binding site residues were experimentally found to block T activation of GPRC6A, in agreement with the modeling. Using Gpr6ca(-/-) mice, we confirmed that loss of GPRC6A resulted in loss of T rapid signaling responses and elucidated several biological functions regulated by GPRC6A-dependent T rapid signaling, including T stimulation of insulin secretion in pancreatic islets and enzyme expression involved in the biosynthesis of T in Leydig cells. Finally, we identified a stereo-specific effect of an R-isomer of a selective androgen receptor modulator that is predicted to bind to and shown to activate GPRC6A but not androgen receptor. Together, our data show that GPRC6A directly mediates the rapid signaling response to T and uncovers previously unrecognized endocrine networks.« less

Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

NASA Astrophysics Data System (ADS)

Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

2015-02-01

The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds

NASA Astrophysics Data System (ADS)

Harada, Shusaku; Hiromori, Youhei; Nakamura, Shota; Kawahara, Kazuki; Fukakusa, Shunsuke; Maruno, Takahiro; Noda, Masanori; Uchiyama, Susumu; Fukui, Kiichi; Nishikawa, Jun-Ichi; Nagase, Hisamitsu; Kobayashi, Yuji; Yoshida, Takuya; Ohkubo, Tadayasu; Nakanishi, Tsuyoshi

2015-02-01

Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT.

Structural basis for PPARγ transactivation by endocrine-disrupting organotin compounds

PubMed Central

Harada, Shusaku; Hiromori, Youhei; Nakamura, Shota; Kawahara, Kazuki; Fukakusa, Shunsuke; Maruno, Takahiro; Noda, Masanori; Uchiyama, Susumu; Fukui, Kiichi; Nishikawa, Jun-ichi; Nagase, Hisamitsu; Kobayashi, Yuji; Yoshida, Takuya; Ohkubo, Tadayasu; Nakanishi, Tsuyoshi

2015-01-01

Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator–activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT. PMID:25687586

Selective binding behavior of humic acid removal by aluminum coagulation.

PubMed

Jin, Pengkang; Song, Jina; Yang, Lei; Jin, Xin; Wang, Xiaochang C

2018-02-01

The reactivity characteristics of humic acid (HA) with aluminium coagulants at different pH values was investigated. It revealed that the linear complexation reaction occurred between aluminum and humic acid at pH < 7, and the reaction rate increased as the pH increased from 2 to 6. While at pH = 7, most of the dosed aluminum existed in the form of free aluminum and remained unreacted in the presence of HA until the concentration reached to trigger Al(OH) 3(s) formation. Differentiating the change of functional groups of HA by 1 H nuclear magnetic resonance spectroscopy and X-ray photoelectron spectra analysis, it elucidated that there was a selective complexation between HA and Al with lower Al dosage at pH 5, which was probably due to coordination of the activated functional groups onto aluminium. While almost all components were removed proportionally by sweep adsorption without selectivity at pH 7, as well as that with higher Al dosage at pH 5. This study provided a promising pathway to analyse the mechanism of the interaction between HA and metal coagulants in future. Copyright © 2017 Elsevier Ltd. All rights reserved.

IFN-λ cancer immunotherapy: new kid on the block

PubMed Central

Lasfar, Ahmed; Gogas, Helen; Zloza, Andrew; Kaufman, Howard L; Kirkwood, John M

2016-01-01

Interferon-lambda (IFN-λ) is a new IFN type, related to IFN-α, that is commonly used in the clinic. However, significant side effects accompanying IFN-α treatment limit enthusiasm for IFN-α. In this review, we discuss the current landscape of IFN-α use in oncology and describe the biologic characteristics of IFN-λ. IFN-λ offers unique advantages, including a more tumor cell selective targeting, lower off-target binding and an ability to generate both innate and adaptive immune responses. IFN-λ has also demonstrated therapeutic benefit in murine cancer models. IFN-λ may be used in clinic as a single agent or in combination with other immunotherapy agents, such as immune checkpoint inhibitors. Further clinical trials will be needed to fully elucidate the potential of this novel agent in oncology. PMID:27381684

Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection

PubMed Central

Zhukovsky, Eugene A.; Morse, Richard J.; Maus, Marcela V.

2016-01-01

To realize the full potential of cancer immunotherapy, the latest generation immunotherapeutics are designed to harness the potent tumor-killing capacity of T cells. Thus, to mobilize T cells, new optimized bispecific antibody (BsAb) designs, enabling efficient polyclonal redirection of cytotoxic activity through binding to CD3 and a Tumor Associated Antigen (TAA) and refined genetically-modified T cells have recently expanded the arsenal of available options for cancer treatment. This review presents the current understanding of the parameters crucial to the design of optimal T cell redirecting BsAb and chimeric antigen receptor (CAR)-modified T cells. However, there are additional questions that require thorough elucidation. Both modalities will benefit from design changes that may increase the therapeutic window. One such approach could employ the discrimination afforded by multiple TAA to significantly increase selectivity. PMID:26963133

Investigation of the binding sites and orientation of caffeine on human serum albumin by surface-enhanced Raman scattering and molecular docking

NASA Astrophysics Data System (ADS)

Wang, Weinan; Zhang, Wei; Duan, Yaokai; Jiang, Yong; Zhang, Liangren; Zhao, Bing; Tu, Pengfei

2013-11-01

Fluorescence, normal Raman and surface-enhanced Raman scattering (SERS) were introduced to explore the absorptive geometry of caffeine on Human Serum Albumin (HSA) at physiological condition. The molecular docking was also employed to make a better understanding of the interaction between caffeine and HSA as well as to elucidate the detailed information of the major binding site. The results showed that caffeine could bind to HSA via the hydrophobic force of aromatic stacking and the main binding group on caffeine could be the pyrimidine ring. In addition, a consecutive set of changes in the orientation of caffeine molecule had been demonstrated during the process of caffeine binding to HSA, and the primary binding site was considered to be a hydrophobic cavity formed by Leu198, Lys199, Ser202, Phe211, Trp214, Val344, Ser454 and Leu481 in domain II.

Peculiarities of binding composition production in vortex jet mill

NASA Astrophysics Data System (ADS)

Zagorodnyuk, L. Kh; Lesovik, V. S.; Sumskoy, D. A.; Elistratkin, M. Yu; Makhortov, D. S.

2018-03-01

The article investigates the disintegration of perlite production waste in a vortex jet mill; the regularities of milling were established. Binding compositions were obtained at different ratios of cement vs. perlite sand production waste in the vortex jet mill in various milling regimes. The peculiarities of milling processes were studied, and technological and physicomechanical properties of the binding compositions were determined as well. The microstructure of the cement stones made of activated Portland cement and binding compositions in the vortex jet mill was elucidated by electron microscopy. The open pores of the cement-binding compositions prepared using perlite fillers were found to be filled by newgrowths at different stages of collective growth. The microstructure of the binding compositions is dense due to rationally proportioned composition, effective mineral filler— perlite waste — that creates additional substrates for internal composite microstructure formation, mechanochemical activation of raw mixture, which allows obtaining composites with required properties.

Synthesis and structure elucidation of a copper(II) Schiff-base complex: in vitro DNA binding, pBR322 plasmid cleavage and HSA binding studies.

PubMed

Tabassum, Sartaj; Ahmad, Musheer; Afzal, Mohd; Zaki, Mehvash; Bharadwaj, Parimal K

2014-11-01

New copper(II) complex with Schiff base ligand 4-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-benzoic acid (H₂L) was synthesized and characterized by spectroscopic and analytical and single crystal X-ray diffraction studies which revealed that the complex 1 exist in a distorted octahedral environment. In vitro CT-DNA binding studies were performed by employing different biophysical technique which indicated that the 1 strongly binds to DNA in comparison to ligand via electrostatic binding mode. Complex 1 cleaves pBR322 DNA via hydrolytic pathway and recognizes minor groove of DNA double helix. The HSA binding results showed that ligand and complex 1 has ability to quench the fluorescence emission intensity of Trp 214 residue available in the subdomain IIA of HSA. Copyright © 2014 Elsevier B.V. All rights reserved.

The ligand binding mechanism to purine nucleoside phosphorylase elucidated via molecular dynamics and machine learning.

PubMed

Decherchi, Sergio; Berteotti, Anna; Bottegoni, Giovanni; Rocchia, Walter; Cavalli, Andrea

2015-01-27

The study of biomolecular interactions between a drug and its biological target is of paramount importance for the design of novel bioactive compounds. In this paper, we report on the use of molecular dynamics (MD) simulations and machine learning to study the binding mechanism of a transition state analogue (DADMe-immucillin-H) to the purine nucleoside phosphorylase (PNP) enzyme. Microsecond-long MD simulations allow us to observe several binding events, following different dynamical routes and reaching diverse binding configurations. These simulations are used to estimate kinetic and thermodynamic quantities, such as kon and binding free energy, obtaining a good agreement with available experimental data. In addition, we advance a hypothesis for the slow-onset inhibition mechanism of DADMe-immucillin-H against PNP. Combining extensive MD simulations with machine learning algorithms could therefore be a fruitful approach for capturing key aspects of drug-target recognition and binding.

Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides.

PubMed

Kalathiya, Umesh; Padariya, Monikaben; Baginski, Maciej

2017-03-01

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1 TRFH -TIN2 TBM and TRF2 TRFH -TIN2 TBM /Apollo TBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1 TRFH and TRF2 TRFH and forming a stable hydrogen bond network with TIN2 TBM or Apollo TBM peptides were traced. Our study revealed that TIN2 TBM forms a well-defined binding mode with TRF1 TRFH as compared to TRF2 TRFH , and that the binding pocket of TIN2 TBM is deeper for TRF2 TRFH protein than Apollo TBM . The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Current drug treatments targeting dopamine D3 receptor.

PubMed

Leggio, Gian Marco; Bucolo, Claudio; Platania, Chiara Bianca Maria; Salomone, Salvatore; Drago, Filippo

2016-09-01

Dopamine receptors (DR) have been extensively studied, but only in recent years they became object of investigation to elucidate the specific role of different subtypes (D1R, D2R, D3R, D4R, D5R) in neural transmission and circuitry. D1-like receptors (D1R and D5R) and D2-like receptors (D2R, D2R and D4R) differ in signal transduction, binding profile, localization in the central nervous system and physiological effects. D3R is involved in a number of pathological conditions, including schizophrenia, Parkinson's disease, addiction, anxiety, depression and glaucoma. Development of selective D3R ligands has been so far challenging, due to the high sequence identity and homology shared by D2R and D3R. As a consequence, despite a rational design of selective DR ligands has been carried out, none of currently available medicines selectively target a given D2-like receptor subtype. The availability of the D3R ligand [(11)C]-(+)-PHNO for positron emission tomography studies in animal models as well as in humans, allows researchers to estimate the expression of D3R in vivo; displacement of [(11)C]-(+)-PHNO binding by concurrent drug treatments is used to estimate the in vivo occupancy of D3R. Here we provide an overview of studies indicating D3R as a target for pharmacological therapy, and a review of market approved drugs endowed with significant affinity at D3R that are used to treat disorders where D3R plays a relevant role. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteotoxic Stress Induces Phosphorylation of p62/SQSTM1 by ULK1 to Regulate Selective Autophagic Clearance of Protein Aggregates

PubMed Central

Lim, Junghyun; Lachenmayer, M. Lenard; Wu, Shuai; Liu, Wenchao; Kundu, Mondira; Wang, Rong; Komatsu, Masaaki; Oh, Young J.; Zhao, Yanxiang; Yue, Zhenyu

2015-01-01

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington’s disease. PMID:25723488

Translating natural genetic variation to gene expression in a computational model of the Drosophila gap gene regulatory network

PubMed Central

Kozlov, Konstantin N.; Kulakovskiy, Ivan V.; Zubair, Asif; Marjoram, Paul; Lawrie, David S.; Nuzhdin, Sergey V.; Samsonova, Maria G.

2017-01-01

Annotating the genotype-phenotype relationship, and developing a proper quantitative description of the relationship, requires understanding the impact of natural genomic variation on gene expression. We apply a sequence-level model of gap gene expression in the early development of Drosophila to analyze single nucleotide polymorphisms (SNPs) in a panel of natural sequenced D. melanogaster lines. Using a thermodynamic modeling framework, we provide both analytical and computational descriptions of how single-nucleotide variants affect gene expression. The analysis reveals that the sequence variants increase (decrease) gene expression if located within binding sites of repressors (activators). We show that the sign of SNP influence (activation or repression) may change in time and space and elucidate the origin of this change in specific examples. The thermodynamic modeling approach predicts non-local and non-linear effects arising from SNPs, and combinations of SNPs, in individual fly genotypes. Simulation of individual fly genotypes using our model reveals that this non-linearity reduces to almost additive inputs from multiple SNPs. Further, we see signatures of the action of purifying selection in the gap gene regulatory regions. To infer the specific targets of purifying selection, we analyze the patterns of polymorphism in the data at two phenotypic levels: the strengths of binding and expression. We find that combinations of SNPs show evidence of being under selective pressure, while individual SNPs do not. The model predicts that SNPs appear to accumulate in the genotypes of the natural population in a way biased towards small increases in activating action on the expression pattern. Taken together, these results provide a systems-level view of how genetic variation translates to the level of gene regulatory networks via combinatorial SNP effects. PMID:28898266

Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor.

PubMed

Kappel, Kalli; Miao, Yinglong; McCammon, J Andrew

2015-11-01

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs.

Preferential Interaction of Na+ over K+ to Carboxylate-functionalized Silver Nanoparticles

EPA Science Inventory

Elucidating mechanistic interactions between specific ions (Na+/ K+) and nanoparticle surfaces to alter particle stability in polar media has received little attention. We investigated relative preferential binding of Na+ and K+ to carboxylate-functionalized silver nanoparticles ...

Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

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

Patterson, Edward I.; EH Graham Centre for Agricultural Innovation; Dombrovski, Andrew K.

2013-09-06

Highlights: •Circovirus capsid proteins contain large nuclear localization signals (NLS). •A method of nuclear import has not been elucidated. •Beak and feather disease virus (BFDV) capsid NLS was crystallized with importin α. •The structure showed BFDV NLS binding to the major site of importin α. •Result shows implications for mechanism of nuclear transport for all circoviruses. -- Abstract: Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediatemore » nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9 Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface.« less

Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis.

PubMed

He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

2016-04-15

The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors.

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

NASA Astrophysics Data System (ADS)

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-07-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment.

Competitive Binding of Natural Amphiphiles with Graphene Derivatives

PubMed Central

Radic, Slaven; Geitner, Nicholas K.; Podila, Ramakrishna; Käkinen, Aleksandr; Chen, Pengyu; Ke, Pu Chun; Ding, Feng

2013-01-01

Understanding the transformation of graphene derivatives by natural amphiphiles is essential for elucidating the biological and environmental implications of this emerging class of engineered nanomaterials. Using rapid discrete-molecular-dynamics simulations, we examined the binding of graphene and graphene oxide with peptides, fatty acids, and cellulose, and complemented our simulations by experimental studies of Raman spectroscopy, FTIR, and UV-Vis spectrophotometry. Specifically, we established a connection between the differential binding and the conformational flexibility, molecular geometry, and hydrocarbon content of the amphiphiles. Importantly, our dynamics simulations revealed a Vroman-like competitive binding of the amphiphiles for the graphene oxide substrate. This study provides a mechanistic basis for addressing the transformation, evolution, transport, biocompatibility, and toxicity of graphene derivatives in living systems and the natural environment. PMID:23881402

Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine.

PubMed

Ohno, Satoshi; Matsui, Megumi; Yokogawa, Takashi; Nakamura, Masashi; Hosoya, Takamitsu; Hiramatsu, Toshiyuki; Suzuki, Masaaki; Hayashi, Nobuhiro; Nishikawa, Kazuya

2007-03-01

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.

Binding of anticancer drug daunomycin to a TGGGGT G-quadruplex DNA probed by all-atom molecular dynamics simulations: additional pure groove binding mode and implications on designing more selective G-quadruplex ligands.

PubMed

Shen, Zhanhang; Mulholland, Kelly A; Zheng, Yujun; Wu, Chun

2017-09-01

DNA G-quadruplex structures are emerging cancer-specific targets for chemotherapeutics. Ligands that bind to and stabilize DNA G-quadruplexes have the potential to be anti-cancer drugs. Lack of binding selectivity to DNA G-quadruplex over DNA duplex remains a major challenge when attempting to develop G-quadruplex ligands into successful anti-cancer drugs. Thorough understanding of the binding nature of existing non-selective ligands that bind to both DNA quadruplex and DNA duplex will help to address this challenge. Daunomycin and doxorubicin, two commonly used anticancer drugs, are examples of non-selective DNA ligands. In this study, we extended our early all-atom binding simulation studies between doxorubicin and a DNA duplex (d(CGATCG) 2 ) to probe the binding between daunomycin and a parallel DNA quadruplex (d(TGGGGT) 4 ) and DNA duplex. In addition to the end stacking mode, which mimics the mode in the crystal structure, a pure groove binding mode was observed in our free binding simulations. The dynamic and energetic properties of these two binding modes are thoroughly examined, and a detailed comparison is made between DNA quadruplex binding modes and DNA duplex binding modes. Implications on the design of more selective DNA quadruplex ligands are also discussed. Graphical abstract Top stacking and groov binding modes from the MD simulations.

Structure-based optimization of oxadiazole-based GSK-3 inhibitors.

PubMed

Lo Monte, Fabio; Kramer, Thomas; Gu, Jiamin; Brodrecht, Martin; Pilakowski, Johannes; Fuertes, Ana; Dominguez, Juan Manuel; Plotkin, Batya; Eldar-Finkelman, Hagit; Schmidt, Boris

2013-03-01

Inhibition of glycogen synthase kinase-3 (GSK-3) induces neuroprotective effects, e.g. decreases β-amyloid production and reduces tau hyperphosphorylation, which are both associated with Alzheimer's disease (AD). The two isoforms of GSK-3 in mammalians are GSK-3α and β, which share 98% homology in their catalytic domains. We investigated GSK-3 inhibitors based on 2 different scaffolds in order to elucidate the demands of the ATP-binding pocket [1]. Particularly, the oxadiazole scaffold provided potent and selective GSK-3 inhibitors. For example, the most potent inhibitor of the present series, the acetamide 26d, is characterized by an IC50 of 2 nM for GSK-3α and 17 nM for GSK-3β. In addition, the benzodioxane 8g showed up to 27-fold selectivity for GSK-3α over GSK-3β, with an IC50 of 35 nM for GSK-3α. Two GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay to evaluate simultaneously permeability and safety. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Structural analysis of malaria-parasite lysyl-tRNA synthetase provides a platform for drug development.

PubMed

Khan, Sameena; Garg, Ankur; Camacho, Noelia; Van Rooyen, Jason; Kumar Pole, Anil; Belrhali, Hassan; Ribas de Pouplana, Lluis; Sharma, Vinay; Sharma, Amit

2013-05-01

Aminoacyl-tRNA synthetases are essential enzymes that transmit information from the genetic code to proteins in cells and are targets for antipathogen drug development. Elucidation of the crystal structure of cytoplasmic lysyl-tRNA synthetase from the malaria parasite Plasmodium falciparum (PfLysRS) has allowed direct comparison with human LysRS. The authors' data suggest that PfLysRS is dimeric in solution, whereas the human counterpart can also adopt tetrameric forms. It is shown for the first time that PfLysRS is capable of synthesizing the signalling molecule Ap4a (diadenosine tetraphosphate) using ATP as a substrate. The PfLysRS crystal structure is in the apo form, such that binding to ATP will require rotameric changes in four conserved residues. Differences in the active-site regions of parasite and human LysRSs suggest the possibility of exploiting PfLysRS for selective inhibition. These investigations on PfLysRS further validate malarial LysRSs as attractive antimalarial targets and provide new structural space for the development of inhibitors that target pathogen LysRSs selectively.

Structure-Based Design of Orally Bioavailable 1H-Pyrrolo[3,2-c]pyridine Inhibitors of Mitotic Kinase Monopolar Spindle 1 (MPS1)

PubMed Central

2013-01-01

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition. PMID:24256217

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

PubMed

Naud, Sébastien; Westwood, Isaac M; Faisal, Amir; Sheldrake, Peter; Bavetsias, Vassilios; Atrash, Butrus; Cheung, Kwai-Ming J; Liu, Manjuan; Hayes, Angela; Schmitt, Jessica; Wood, Amy; Choi, Vanessa; Boxall, Kathy; Mak, Grace; Gurden, Mark; Valenti, Melanie; de Haven Brandon, Alexis; Henley, Alan; Baker, Ross; McAndrew, Craig; Matijssen, Berry; Burke, Rosemary; Hoelder, Swen; Eccles, Suzanne A; Raynaud, Florence I; Linardopoulos, Spiros; van Montfort, Rob L M; Blagg, Julian

2013-12-27

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Adsorption and Reaction of Acetaldehyde on Shape-Controlled CeO2 Nanocrystals: Elucidation of Structure-function Relationships

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

Mann, Amanda K; Wu, Zili; Calaza, Florencia

2014-01-01

CeO2 cubes with {100} facets, octahedra with {111} facets, and wires with highly defective structures were utilized to probe the structure-dependent reactivity of acetaldehyde. Using temperature-programmed desorption (TPD), temperature-programmed surface reactions (TPSR), and in situ infrared spectroscopy it was found that acetaldehyde desorbs unreacted or undergoes reduction, coupling, or C-C bond scission reactions depending on the surface structure of CeO2. Room temperature FTIR indicates that acetaldehyde binds primarily as 1-acetaldehyde on the octahedra, in a variety of conformations on the cubes, including coupling products and acetate and enolate species, and primarily as coupling products on the wires. The percent consumptionmore » of acetaldehyde follows the order of wires > cubes > octahedra. All the nanoshapes produce the coupling product crotonaldehyde; however, the selectivity to produce ethanol follows the order wires cubes >> octahedra. The selectivity and other differences can be attributed to the variation in the basicity of the surfaces, defects densities, coordination numbers of surface atoms, and the reducibility of the nanoshapes.« less

Computational methods for analysis and inference of kinase/inhibitor relationships

PubMed Central

Ferrè, Fabrizio; Palmeri, Antonio; Helmer-Citterich, Manuela

2014-01-01

The central role of kinases in virtually all signal transduction networks is the driving motivation for the development of compounds modulating their activity. ATP-mimetic inhibitors are essential tools for elucidating signaling pathways and are emerging as promising therapeutic agents. However, off-target ligand binding and complex and sometimes unexpected kinase/inhibitor relationships can occur for seemingly unrelated kinases, stressing that computational approaches are needed for learning the interaction determinants and for the inference of the effect of small compounds on a given kinase. Recently published high-throughput profiling studies assessed the effects of thousands of small compound inhibitors, covering a substantial portion of the kinome. This wealth of data paved the road for computational resources and methods that can offer a major contribution in understanding the reasons of the inhibition, helping in the rational design of more specific molecules, in the in silico prediction of inhibition for those neglected kinases for which no systematic analysis has been carried yet, in the selection of novel inhibitors with desired selectivity, and offering novel avenues of personalized therapies. PMID:25071826

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin

PubMed Central

Westerlund, Annie M.

2018-01-01

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity. PMID:29614072

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

PubMed

Westerlund, Annie M; Delemotte, Lucie

2018-04-01

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

Entropy as a Driver of Selectivity for Inhibitor Binding to Histone Deacetylase 6.

PubMed

Porter, Nicholas J; Wagner, Florence F; Christianson, David W

2018-05-18

Among the metal-dependent histone deacetylases, the class IIb isozyme HDAC6 is remarkable because of its role in the regulation of microtubule dynamics in the cytosol. Selective inhibition of HDAC6 results in microtubule hyperacetylation, leading to cell cycle arrest and apoptosis, which is a validated strategy for cancer chemotherapy and the treatment of other disorders. HDAC6 inhibitors generally consist of a Zn 2+ -binding group such as a hydroxamate, a linker, and a capping group; the capping group is a critical determinant of isozyme selectivity. Surprisingly, however, even "capless" inhibitors exhibit appreciable HDAC6 selectivity. To probe the chemical basis for this selectivity, we now report high-resolution crystal structures of HDAC6 complexed with capless cycloalkyl hydroxamate inhibitors 1-4. Each inhibitor hydroxamate group coordinates to the catalytic Zn 2+ ion with canonical bidentate geometry. Additionally, the olefin moieties of compounds 2 and 4 bind in an aromatic crevice between the side chains of F583 and F643. Reasoning that similar binding could be achieved in the representative class I isozyme HDAC8, we employed isothermal titration calorimetry to study the thermodynamics of inhibitor binding. These measurements indicate that the entropy of inhibitor binding is generally positive for binding to HDAC6 and negative for binding to HDAC8, resulting in ≤313-fold selectivity for binding to HDAC6 relative to HDAC8. Thus, favorable binding entropy contributes to HDAC6 selectivity. Notably, cyclohexenyl hydroxamate 2 represents a promising lead for derivatization with capping groups that may further enhance its impressive 313-fold thermodynamic selectivity for HDAC6 inhibition.

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.

Profiling charge complementarity and selectivity for binding at the protein surface.

PubMed

Sulea, Traian; Purisima, Enrico O

2003-05-01

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins.

In silico identification and characterization of common epitope-based peptide vaccine for Nipah and Hendra viruses.

PubMed

Saha, Chayan Kumar; Mahbub Hasan, Md; Saddam Hossain, Md; Asraful Jahan, Md; Azad, Abul Kalam

2017-06-01

To explore a common B- and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV). Membrane proteins F, G and M of HeV and NiV were retrieved from the protein database and subjected to different bioinformatics tools to predict antigenic B-cell epitopes. Best B-cell epitopes were then analyzed to predict their T-cell antigenic potentiality. Antigenic B- and T-cell epitopes that shared maximum identity with HeV and NiV were selected. Stability of the selected epitopes was predicted. Finally, the selected epitopes were subjected to molecular docking simulation with HLA-DR to confirm their antigenic potentiality in silico. One epitope from G proteins, one from M proteins and none from F proteins were selected based on their antigenic potentiality. The epitope from the G proteins was stable whereas that from M was unstable. The M-epitope was made stable by adding flanking dipeptides. The 15-mer G-epitope (VDPLRVQWRNNSVIS) showed at least 66% identity with all NiV and HeV G protein sequences, while the 15-mer M-epitope (GKLEFRRNNAIAFKG) with the dipeptide flanking residues showed 73% identity with all NiV and HeV M protein sequences available in the database. Molecular docking simulation with most frequent MHC class-II (MHC II) and class-I (MHC I) molecules showed that these epitopes could bind within HLA binding grooves to elicit an immune response. Data in our present study revealed the notion that the epitopes from G and M proteins might be the target for peptide-based subunit vaccine design against HeV and NiV. However, the biochemical analysis is necessary to experimentally validate the interaction of epitopes individually with the MHC molecules through elucidation of immunity induction. Copyright © 2017 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

The High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects on CD44 Clustering*

PubMed Central

Yang, Cuixia; Cao, Manlin; Liu, Hua; He, Yiqing; Xu, Jing; Du, Yan; Liu, Yiwen; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

2012-01-01

CD44 is a major cell surface receptor for the glycosaminoglycan hyaluronan (HA). Native high molecular weight hyaluronan (nHA) and oligosaccharides of hyaluronan (oHA) provoke distinct biological effects upon binding to CD44. Despite the importance of such interactions, however, the feature of binding with CD44 at the cell surface and the molecular basis for functional distinction between different sizes of HA is still unclear. In this study we investigated the effects of high and low molecular weight hyaluronan on CD44 clustering. For the first time, we provided direct evidence for a strong relationship between HA size and CD44 clustering in vivo. In CD44-transfected COS-7 cells, we showed that exogenous nHA stimulated CD44 clustering, which was disrupted by oHA. Moreover, naturally expressed CD44 was distributed into clusters due to abundantly expressed nHA in HK-2 cells (human renal proximal tubule cells) and BT549 cells (human breast cancer cell line) without exogenous stimulation. Our results suggest that native HA binding to CD44 selectively induces CD44 clustering, which could be inhibited by oHA. Finally, we demonstrated that HA regulates cell adhesion in a manner specifically dependent on its size. oHA promoted cell adhesion while nHA showed no effects. Our results might elucidate a molecular- and/or cellular-based mechanism for the diverse biological activities of nHA and oHA. PMID:23118219

TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116.

PubMed

Choi, Jee-Hye; Min, Na Young; Park, Jina; Kim, Jin Hong; Park, Soo Hyun; Ko, Young Jong; Kang, Yoonsung; Moon, Young Joon; Rhee, Sangmyung; Ham, Seung Wook; Park, Ae Ja; Lee, Kwang-Ho

2010-01-01

Trichostatin A (TSA), an inhibitor of histone deacetylase, is a well-known antitumor agent that effectively and selectively induces tumor growth arrest and apoptosis. Recently, it was reported that hTERT is one of the primary targets for TSA-induced apoptosis in cancer cells but the mechanism of which has not yet been elucidated. In the present study, to better understand the epigenetic regulation mechanism responsible for the repression of hTERT by TSA, we examined expression of hTERT in the HCT116 colon cancer cell line after treatment with TSA and performed site-specific CpG methylation analysis of the hTERT promoter. We found that TSA-induced the demethylation of site-specific CpGs on the promoter of hTERT, which was caused by down-regulation of DNA methyltransferase 1 (DNMT1). Among the demethylated region, the 31st-33rd CpGs contained a binding site for CTCF, an inhibitor of hTERT transcription. ChIP analysis revealed that TSA-induced demethylation of the 31st-33rd CpGs promoted CTCF binding on hTERT promoter, leading to repression of hTERT. Taken together, down-regulation of DNMT1 by TSA caused demethylation of a CTCF binding site on the hTERT promoter, the result of which was repression of hTERT via recruitment of CTCF to the promoter. Copyright 2009 Elsevier Inc. All rights reserved.

Synthesis, characterization and target protein binding of drug-conjugated quantum dots in vitro and in living cells

NASA Astrophysics Data System (ADS)

Choi, Youngseon; Kim, Minjung; Cho, Yoojin; Yun, Eunsuk; Song, Rita

2013-02-01

Elucidation of unknown target proteins of a drug is of great importance in understanding cell biology and drug discovery. There have been extensive studies to discover and identify target proteins in the cell. Visualization of targets using drug-conjugated probes has been an important approach to gathering mechanistic information of drug action at the cellular level. As quantum dot (QD) nanocrystals have attracted much attention as a fluorescent probe in the bioimaging area, we prepared drug-conjugated QD to explore the potential of target discovery. As a model drug, we selected a well-known anticancer drug, methotrexate (MTX), which has been known to target dihydrofolate reductase (DHFR) with high affinity binding (Kd = 0.54 nM). MTX molecules were covalently attached to amino-PEG-polymer-coated QDs. Specific interactions of MTX-conjugated QDs with DHFR were identified using agarose gel electrophoresis and fluorescence microscopy. Cellular uptake of the MTX-conjugated QDs in living CHO cells was investigated with regard to their localization and distribution pattern. MTX-QD was found to be internalized into the cells via caveolae-medicated endocytosis without significant sequestration in endosomes. A colocalization experiment of the MTX-QD conjugate with antiDHFR-TAT-QD also confirmed that MTX-QD binds to the target DHFR. This study showed the potential of the drug-QD conjugate to identify or visualize drug-target interactions in the cell, which is currently of great importance in the area of drug discovery and chemical biology.

In Silico Detection of Sequence Variations Modifying Transcriptional Regulation

PubMed Central

Andersen, Malin C; Engström, Pär G; Lithwick, Stuart; Arenillas, David; Eriksson, Per; Lenhard, Boris; Wasserman, Wyeth W; Odeberg, Jacob

2008-01-01

Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers). The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation. PMID:18208319

Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation.

PubMed

Bochicchio, Anna; Jordaan, Sandra; Losasso, Valeria; Chetty, Shivan; Perera, Rodrigo Casasnovas; Ippoliti, Emiliano; Barth, Stefan; Carloni, Paolo

2017-02-17

Targeted human cytolytic fusion proteins (hCFPs) are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv) specifically binding to e.g., tumor associated antigens (TAAs). After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to upregulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively), reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD) simulations and enhanced sampling methods (ESM). MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such "high-resolution" detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields.

Plasma Levels of Fatty Acid-Binding Protein 4, Retinol-Binding Protein 4, High-Molecular Weight Adiponectin, and Cardiovascular Mortality among Men with Type 2 Diabetes: A 22-Year Prospective Study

PubMed Central

Liu, Gang; Ding, Ming; Chiuve, Stephanie E.; Rimm, Eric B.; Franks, Paul W.; Meigs, James B.; Hu, Frank B.; Sun, Qi

2016-01-01

Objective To examine select adipokines, including fatty acid-binding protein 4 (FABP4), retinol-binding protein 4 (RBP4), and high-molecular weight (HMW) adiponectin in relation to cardiovascular disease (CVD) mortality among patients with type 2 diabetes (T2D). Approach and Results Plasma levels of FABP4, RBP4, and HMW adiponectin were measured in 950 men with T2D in the Health Professionals Follow-up Study. After an average of 22 years of follow up (1993–2015), 580 deaths occurred, of whom 220 died of CVD. After multivariate adjustment for covariates, higher levels of FABP4 were significantly associated with a higher CVD mortality: comparing extreme tertiles, the hazard ratio (HR) and 95% confidence interval (CI) of CVD mortality was 1.78 (1.22, 2.59; P trend=0.001). A positive association was also observed for HMW adiponectin: the HR (95% CI) was 2.07 (1.42, 3.06; P trend=0.0002), comparing extreme tertiles, whereas higher RBP4 levels were non-significantly associated with a decreased CVD mortality with an HR (95% CI) of 0.73 (0.50, 1.07; P trend=0.09). A Mendelian randomization (MR) analysis suggested that the causal relationships of HMW adiponectin and RBP4 would be directionally opposite to those observed based on the biomarkers, although none of the MR associations achieved statistical significance. Conclusions These data suggest that higher levels of FABP4 and HMW adiponectin are associated with elevated CVD mortality among men with T2D. Biological mechanisms underlying these observations deserve elucidation, but the associations of HMW adiponectin may partially reflect altered adipose tissue functionality among T2D patients. PMID:27609367

Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation

PubMed Central

Bochicchio, Anna; Jordaan, Sandra; Losasso, Valeria; Chetty, Shivan; Casasnovas Perera, Rodrigo; Ippoliti, Emiliano; Barth, Stefan; Carloni, Paolo

2017-01-01

Targeted human cytolytic fusion proteins (hCFPs) are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv) specifically binding to e.g., tumor associated antigens (TAAs). After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to up-regulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively), reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD) simulations and enhanced sampling methods (ESM). MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such “high-resolution” detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields. PMID:28536352

Amino acid signature enables proteins to recognize modified tRNA.

PubMed

Spears, Jessica L; Xiao, Xingqing; Hall, Carol K; Agris, Paul F

2014-02-25

Human tRNA(Lys3)UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNA(Lys3)UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNA(Lys3)UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N(6)-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNA(Lys3)UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA's fully modified anticodon stem and loop domain, hASL(Lys3)UUU. Peptides of this sequence specifically recognized and bound modified htRNA(Lys3)UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

2013-11-01

Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Binding ligand prediction for proteins using partial matching of local surface patches.

PubMed

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group.

Binding Ligand Prediction for Proteins Using Partial Matching of Local Surface Patches

PubMed Central

Sael, Lee; Kihara, Daisuke

2010-01-01

Functional elucidation of uncharacterized protein structures is an important task in bioinformatics. We report our new approach for structure-based function prediction which captures local surface features of ligand binding pockets. Function of proteins, specifically, binding ligands of proteins, can be predicted by finding similar local surface regions of known proteins. To enable partial comparison of binding sites in proteins, a weighted bipartite matching algorithm is used to match pairs of surface patches. The surface patches are encoded with the 3D Zernike descriptors. Unlike the existing methods which compare global characteristics of the protein fold or the global pocket shape, the local surface patch method can find functional similarity between non-homologous proteins and binding pockets for flexible ligand molecules. The proposed method improves prediction results over global pocket shape-based method which was previously developed by our group. PMID:21614188

Is the isolated ligand binding domain a good model of the domain in the native receptor?

PubMed

Deming, Dustin; Cheng, Qing; Jayaraman, Vasanthi

2003-05-16

Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.

Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations.

PubMed

van der Vaart, Arjan

2015-05-01

Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus.

PubMed

Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

2012-01-01

Thioredoxin binding protein -2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein -2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein -2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein -2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein -2 in metabolic control. Enhancement of thioredoxin binding protein -2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein -2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein -2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β(2)-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus.

Urban Biomining: Biological Extraction of Metals and Materials from Electronics Waste Using a Synthetic Biology Approach

NASA Astrophysics Data System (ADS)

Urbina-Navarrete, J.; Rothschild, L.

2016-12-01

End-of-life electronics waste (e-waste) containing toxic and valuable materials is a rapidly progressing human health and environmental issue. Using synthetic biology tools, we have developed a recycling method for e-waste. Our innovation is to use a recombinant version of a naturally-occurring silica-degrading enzyme to depolymerize the silica in metal- and glass- containing e-waste components, and subsequently, to use engineered bacterial surfaces to bind and separate metals from a solution. The bacteria with bound metals can then be used as "bio-ink" to print new circuits using a novel plasma jet electronics printing technology. Here, we present the results from our initial studies that focus on the specificity of metal-binding motifs for a cognate metal. The candidate motifs that show high affinity and specificity will be engineered into bacterial surfaces for downstream applications in biologically-mediated metal recycling. Since the chemistry and role of Cu in metalloproteins is relatively well-characterized, we are using Cu as a proxy to elucidate metal and biological ligand interactions with various metals in e-waste. We assess the binding parameters of 3 representative classes of Cu-binding motifs using isothermal titration calorimetry; 1) natural motifs found in metalloproteins, 2) consensus motifs, and 3) rationally designed peptides that are predicted, in silico, to bind Cu. Our results indicate that naturally-occurring motifs have relative high affinity and specificity for Cu (association constant for Cu Ka 104 M-1, Zn Ka 103 M-1) when competing ions are present in the aqueous milieu. However, motifs developed through rational design by applying quantum mechanical methods that take into account complexation energies of the elemental binding partners and molecular geometry of the cognate metal, not only show high affinity for the cognate metal (Cu Ka 106 M-1), but they show specificity and discrimination against other metal ions that would be competitors for the same binding sites. This is an initial proof-of-concept study that focuses on Cu-binding; however the overall objective of this research is to have peptides that selectively bind many metals from e-waste and this would allow for the separation of the metals from a solution, at ambient temperatures and under non-toxic conditions.

Dyes as bifunctional markers of DNA hybridization on surfaces and mutation detection.

PubMed

García-Mendiola, Tania; Cerro, María Ramos; López-Moreno, José María; Pariente, Félix; Lorenzo, Encarnación

2016-10-01

The interaction of small molecules with DNA has found diagnostic and therapeutic applications. In this work, we propose the use of two different dyes, in particular Azure A and Safranine, as bifunctional markers of on-surface DNA hybridization and potent tools for screening of specific gene mutations directly in real DNA PCR amplicons extracted from blood cells. By combining spectroscopic and electrochemical methods we demonstrate that both dyes can interact with single and double stranded DNA to a different extent, allowing reliable hybridization detection. From these data, we have also elucidated the nature of the interaction. We conclude that the binding mode is fundamentally intercalative with an electrostatic component. The dye fluorescence allows their use as nucleic acid stains for the detection of on-surfaces DNA hybridization. Its redox activity is exploited in the development of selective electrochemical DNA biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Discovery and Pre-Clinical Characterization of Third-Generation 4-H Heteroaryldihydropyrimidine (HAP) Analogues as Hepatitis B Virus (HBV) Capsid Inhibitors.

PubMed

Qiu, Zongxing; Lin, Xianfeng; Zhang, Weixing; Zhou, Mingwei; Guo, Lei; Kocer, Buelent; Wu, Guolong; Zhang, Zhisen; Liu, Haixia; Shi, Houguang; Kou, Buyu; Hu, Taishan; Hu, Yimin; Huang, Mengwei; Yan, S Frank; Xu, Zhiheng; Zhou, Zheng; Qin, Ning; Wang, Yue Fen; Ren, Shuang; Qiu, Hongxia; Zhang, Yuxia; Zhang, Yi; Wu, Xiaoyue; Sun, Kai; Zhong, Sheng; Xie, Jianxun; Ottaviani, Giorgio; Zhou, Yuan; Zhu, Lina; Tian, Xiaojun; Shi, Liping; Shen, Fang; Mao, Yi; Zhou, Xue; Gao, Lu; Young, John A T; Wu, Jim Zhen; Yang, Guang; Mayweg, Alexander V; Shen, Hong C; Tang, Guozhi; Zhu, Wei

2017-04-27

Described herein are the discovery and structure-activity relationship (SAR) studies of the third-generation 4-H heteroaryldihydropyrimidines (4-H HAPs) featuring the introduction of a C6 carboxyl group as novel HBV capsid inhibitors. This new series of 4-H HAPs showed improved anti-HBV activity and better drug-like properties compared to the first- and second-generation 4-H HAPs. X-ray crystallographic study of analogue 12 (HAP_R01) with Cp149 Y132A mutant hexamer clearly elucidated the role of C6 carboxyl group played for the increased binding affinity, which formed strong hydrogen bonding interactions with capsid protein and coordinated waters. The representative analogue 10 (HAP_R10) was extensively characterized in vitro (ADMET) and in vivo (mouse PK and PD) and subsequently selected for further development as oral anti-HBV infection agent.

[Studies on the structure-activity relationship of retinoids--Hansch analysis and 3D-OSAR studies on specific ligands of retinoid x receptor].

PubMed

Huang, N; Chu, F; Guo, Z

1998-06-01

Retinoids (Vitamin A, its metabolites and synthetic analogues) play important roles in a variety of biological processes, including cellular differentiation, proliferation and apoptosis. The many diverse actions of retinoids attribute to the ability of regulating transcription of different target genes through activation of multiple retinoid nuclear receptors (RAR of RXR). So, retinoids with selective binding ability to specific receptor may not only have improved therapeutic indices, but may also be invaluable for elucidating the molecular mechanism of retinoidal transcriptional activation. Based on the two dimensional and three dimensional quantitative structure-activity relationships of specific ligands of RXR, we carried out mimesis of environment of ligands interacting with their receptor and, to some extent, mapping the topological and physico-chemical characteristics of receptor. The knowledge of the QSAR study will offer detailed molecular information for design, synthesis and biological evaluation in drug research and development.

The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis

PubMed Central

van der Westhuyzen, Renier; Hammons, Justin C.; Meier, Jordan L.; Dahesh, Samira; Moolman, Wessel J. A.; Pelly, Stephen C.; Nizet, Victor; Burkart, Michael D.; Strauss, Erick

2012-01-01

SUMMARY The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics, and highlight CoA biosynthesis as a viable antimicrobial drug target. PMID:22633408

GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana.

PubMed

Sawa, Mariko; Kay, Steve A

2011-07-12

Plants perceive environmental signals such as day length and temperature to determine optimal timing for the transition from vegetative to floral stages. Arabidopsis flowers under long-day conditions through the CONSTANS (CO)-FLOWERING LOCUS T (FT) regulatory module. It is thought that the environmental cues for photoperiodic control of flowering are initially perceived in the leaves. We have previously shown that GIGANTEA (GI) regulates the timing of CO expression, together with FLAVIN-BINDING, KELCH REPEAT, F BOX protein 1. Normally, CO and FT are expressed exclusively in vascular bundles, whereas GI is expressed in various tissues. To better elucidate the role of tissue-specific expression of GI in the flowering pathway, we established transgenic lines in which GI is expressed exclusively in mesophyll, vascular bundles, epidermis, shoot apical meristem, or root. We found that GI expressed in either mesophyll or vascular bundles rescues the late-flowering phenotype of the gi-2 loss-of-function mutant under both short-day and long-day conditions. Interestingly, GI expressed in mesophyll or vascular tissues increases FT expression without up-regulating CO expression under short-day conditions. Furthermore, we examined the interaction between GI and FT repressors in mesophyll. We found that GI can bind to three FT repressors: SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO (TEM)1, and TEM2. Finally, our chromatin immunoprecipitation experiments showed that GI binds to FT promoter regions that are near the SVP binding sites. Taken together, our data further elucidate the multiple roles of GI in the regulation of flowering time.

Binding of indomethacin methyl ester to cyclooxygenase-2. A computational study.

PubMed

Sárosi, Menyhárt-Botond

2018-06-05

Inhibitors selective towards the second isoform of prostaglandin synthase (cyclooxygenase, COX-2) are promising nonsteroidal anti-inflammatory drugs and antitumor medications. Methylation of the carboxylate group in the relatively nonselective COX inhibitor indomethacin confers significant COX-2 selectivity. Several other modifications converting indomethacin into a COX-2 selective inhibitor have been reported. Earlier experimental and computational studies on neutral indomethacin derivatives suggest that the methyl ester derivative likely binds to COX-2 with a similar binding mode as that observed for the parent indomethacin. However, docking studies followed by molecular dynamics simulations revealed two possible binding modes in COX-2 for indomethacin methyl ester, which differs from the experimental binding mode found for indomethacin. Both alternative binding modes might explain the observed COX-2 selectivity of indomethacin methyl ester. Graphical abstract Binding of indomethacin methyl ester to cyclooxygenase-2.

MOLECULAR PROCESSES IN CELLULAR ARSENIC METABOLISM

EPA Science Inventory

Elucidating molecular processes that underlie accumulation, metabolism, and binding of iAs and its methylated metabolites provides a basis for understanding the modes of action by which iAs acts as a toxin and a carcinogen. One approach to this problem is to construct a conceptu...

Elimination of Endogenous Toxin, Creatinine from Blood Plasma Depends on Albumin Conformation: Site Specific Uremic Toxicity & Impaired Drug Binding

PubMed Central

Varshney, Ankita; Rehan, Mohd; Subbarao, Naidu; Rabbani, Gulam; Khan, Rizwan Hasan

2011-01-01

Uremic syndrome results from malfunctioning of various organ systems due to the retention of uremic toxins which, under normal conditions, would be excreted into the urine and/or metabolized by the kidneys. The aim of this study was to elucidate the mechanisms underlying the renal elimination of uremic toxin creatinine that accumulate in chronic renal failure. Quantitative investigation of the plausible correlations was performed by spectroscopy, calorimetry, molecular docking and accessibility of surface area. Alkalinization of normal plasma from pH 7.0 to 9.0 modifies the distribution of toxin in the body and therefore may affect both the accumulation and the rate of toxin elimination. The ligand loading of HSA with uremic toxin predicts several key side chain interactions of site I that presumably have the potential to impact the specificity and impaired drug binding. These findings provide useful information for elucidating the complicated mechanism of toxin disposition in renal disease state. PMID:21386972

Elimination of endogenous toxin, creatinine from blood plasma depends on albumin conformation: site specific uremic toxicity & impaired drug binding.

PubMed

Varshney, Ankita; Rehan, Mohd; Subbarao, Naidu; Rabbani, Gulam; Khan, Rizwan Hasan

2011-02-28

Uremic syndrome results from malfunctioning of various organ systems due to the retention of uremic toxins which, under normal conditions, would be excreted into the urine and/or metabolized by the kidneys. The aim of this study was to elucidate the mechanisms underlying the renal elimination of uremic toxin creatinine that accumulate in chronic renal failure. Quantitative investigation of the plausible correlations was performed by spectroscopy, calorimetry, molecular docking and accessibility of surface area. Alkalinization of normal plasma from pH 7.0 to 9.0 modifies the distribution of toxin in the body and therefore may affect both the accumulation and the rate of toxin elimination. The ligand loading of HSA with uremic toxin predicts several key side chain interactions of site I that presumably have the potential to impact the specificity and impaired drug binding. These findings provide useful information for elucidating the complicated mechanism of toxin disposition in renal disease state.

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

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

Wu, Kailang; Peng, Guiqing; Wilken, Matthew

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional,more » and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.« less

A Direct Comparison of the MM-GB/SA Scoring Procedure and Free-Energy Perturbation Calculations Using Carbonic Anhydrase as a Test Case: Strengths and Pitfalls of Each Approach.

PubMed

Guimarães, Cristiano R W

2011-07-12

MM-GB/SA scoring and free energy perturbation (FEP) calculations have emerged as reliable methodologies to understand structural and energetic relationships to binding. In spite of successful applications to elucidate the structure-activity relationships for few pairs of ligands, the reality is that the performance of FEP calculations has rarely been tested for more than a handful of compounds. In this work, a series of 13 benzene sulfonamide inhibitors of carbonic anhydrase with binding free energies determined by isothermal titration calorimetry was selected as a test case. R(2) values of 0.70, 0.71, and 0.49 with the experiment were obtained with MM-GB/SA and FEP simulations run with MCPRO+ and Desmond, respectively. All methods work well, but the results obtained with Desmond are inferior to MM-GB/SA and MCPRO+. The main contrast between the methods is the level of sampling, ranging from full to restricted flexibility to single conformation for the complexes in Desmond, MCPRO+, and MM-GB/SA, respectively. The current and historical results obtained with MM-GB/SA qualify this approach as a more attractive alternative for rank-ordering; it can achieve equivalent or superior predictive accuracy and handle more structurally dissimilar ligands at a fraction of the computational cost of the rigorous free-energy methods. As for the large theoretical dynamic range for the binding energies, that seems to be a direct result of the degree of sampling in the simulations since MCPRO+ as well as MM-GB/SA are plagued by this. Van't Hoff analysis for selected pairs of ligands suggests that the wider scoring spread is not only affected by missing entropic contributions due to restricted sampling but also exaggerated enthalpic separation between the weak and potent compounds caused by diminished shielding of electrostatic interactions, thermal effects, and protein relaxation/strain.

Functionalized Ergot-alkaloids as potential dopamine D3 receptor agonists for treatment of schizophrenia

NASA Astrophysics Data System (ADS)

Ivanova, Bojidarka; Spiteller, Michael

2012-12-01

The relationship between the molecular structure and physical properties of functionalized naturally occurred Ergot-alkaloids as potential dopamine D3 receptor agonists is presented. The molecular modeling of the ergoline-skeleton is based on the comprehensive theoretical study of the binding affinity of the isolated chemicals towards the active sites of the D3 sub-type receptor (D3R) loops. The studied proton accepting ability under physiological conditions allows classifying four types of monocationics, characterizing with the different binding modes to D3R involving selected amino acid residues to the active sites. These results marked the pharmaceutical potential and clinical usage of the reported compounds as antipsychotic drugs for Schizophrenia treatment, since they allowed evaluating the highlights of the different hypothesizes of the biochemical causes the illness. The applied complex approach for theoretical and experimental elucidation, including quantum chemistry method, electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometric (MS) methods, nuclear magnetic resonance and vibrational IR and Raman spectroscopy on the isolated fifteen novel derivatives (1)-(15) and their different protonated forms (1a)-(15a) evidenced a strong dependence of molecular conformation, physical properties and binding affinity. Thus, the semi-synthetic functionalization of the naturally occurred products (NPs), provided significant possibilities to further molecular drugs-design and development of novel derivatives with wanted biological function, using the established profile of selected classes/families of NPs. The work described chiefly the non-linear (NL) approach for the interpretation of the mass chromatograms on the performed hybrid high performance liquid chromatography (HPLC) tandem MS/MS and MS/MS/MS experiments, discussing the merits and great diversity of instrumentation flexibility, thus achieving fundamental structural information, indispensable for the analysis of Ergot-alkaloid derivatives, which under the physiological conditions easily converted to d-lysergic acid (LSD).

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin*

PubMed Central

Chan, Linda J.; Rosengren, K. Johan; Layfield, Sharon L.; Bathgate, Ross A. D.; Separovic, Frances; Samuel, Chrishan S.; Hossain, Mohammed A.; Wade, John D.

2012-01-01

Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4–24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin. PMID:23024363

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus*

PubMed Central

Wu, Kailang; Peng, Guiqing; Wilken, Matthew; Geraghty, Robert J.; Li, Fang

2012-01-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals. PMID:22291007

A selective peroxisome proliferator-activated receptor-gamma modulator, telmisartan, binds to the receptor in a different fashion from thiazolidinediones.

PubMed

Tagami, Tetsuya; Yamamoto, Hiroyuki; Moriyama, Kenji; Sawai, Kuniko; Usui, Takeshi; Shimatsu, Akira; Naruse, Mitsuhide

2009-02-01

Angiotensin type 1 receptor blockers are widely used for the treatment of hypertension, and one angiotensin type 1 receptor blocker, telmisartan, specifically activates the peroxisome proliferator-activated receptor (PPAR)-gamma. We studied the impact of PPARgamma mutants on transcriptional control and interaction with cofactors to elucidate differences in the molecular mechanism between telmisartan and other PPARgamma agonists, thiazolidinediones (TZDs). We created several amino acid substitutions in the ligand binding domain of PPARgamma that, based on molecular modeling, may affect the binding of these agents. In transient expression experiments, wild-type PPARgamma-mediated transcription stimulated by telmisartan was more than one third of that stimulated by TZDs. The activation stimulated by TZDs was impaired, whereas activation stimulated by telmisartan was retained, in the H323Y, S342A, and H449A mutants. In the Y473A mutant, the TZD-induced activation was further impaired and lower than that of telmisartan-induced activation. Coexpression of coactivators enhanced the activation by both telmisartan and TZDs, but activation by telmisartan always exceeded that of TZDs in the Y473A mutant. Based on a mammalian two-hybrid assay, the interaction with corepressors was retained in Y473A. Telmisartan and TZDs, but not 9cis retinoic acid, dissociated corepressors from the wild-type PPARgamma. Telmisartan most effectively dissociated corepressors from Y473A. The interaction with coactivators was enhanced by TZD activation of wild-type PPARgamma and both telmisartan and TZD activation of Y473A. Thus, the Y473A mutant is selectively stimulated by telmisartan but not TZDs, suggesting that telmisartan and TZDs have differential effects on the transcriptional control. In conclusion, these PPARgamma mutants could be powerful tools for developing novel therapeutic agents that retain the metabolic efficacy of PPARgamma activation with fewer adverse effects, such as the increase in body weight associated with TZDs.

Identification of an allosteric binding site for RORγt inhibition

PubMed Central

Scheepstra, Marcel; Leysen, Seppe; van Almen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc

2015-01-01

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors. PMID:26640126

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

Structure-Templated Predictions of Novel Protein Interactions from Sequence Information

PubMed Central

Betel, Doron; Breitkreuz, Kevin E; Isserlin, Ruth; Dewar-Darch, Danielle; Tyers, Mike; Hogue, Christopher W. V

2007-01-01

The multitude of functions performed in the cell are largely controlled by a set of carefully orchestrated protein interactions often facilitated by specific binding of conserved domains in the interacting proteins. Interacting domains commonly exhibit distinct binding specificity to short and conserved recognition peptides called binding profiles. Although many conserved domains are known in nature, only a few have well-characterized binding profiles. Here, we describe a novel predictive method known as domain–motif interactions from structural topology (D-MIST) for elucidating the binding profiles of interacting domains. A set of domains and their corresponding binding profiles were derived from extant protein structures and protein interaction data and then used to predict novel protein interactions in yeast. A number of the predicted interactions were verified experimentally, including new interactions of the mitotic exit network, RNA polymerases, nucleotide metabolism enzymes, and the chaperone complex. These results demonstrate that new protein interactions can be predicted exclusively from sequence information. PMID:17892321

Autoinhibition of ankyrin-B/G membrane target bindings by intrinsically disordered segments from the tail regions

PubMed Central

Wang, Chao; Wei, Zhiyi

2017-01-01

Ankyrins together with their spectrin partners are the master organizers of micron-scale membrane domains in diverse tissues. The 24 ankyrin (ANK) repeats of ankyrins bind to numerous membrane proteins, linking them to spectrin-based cytoskeletons at specific membrane microdomains. The accessibility of the target binding groove of ANK repeats must be regulated to achieve spatially defined functions of ankyrins/target complexes in different tissues, though little is known in this regard. Here we systemically investigated the autoinhibition mechanism of ankyrin-B/G by combined biochemical, biophysical and structural biology approaches. We discovered that the entire ANK repeats are inhibited by combinatorial and quasi-independent bindings of multiple disordered segments located in the ankyrin-B/G linkers and tails, suggesting a mechanistic basis for differential regulations of membrane target bindings by ankyrins. In addition to elucidating the autoinhibition mechanisms of ankyrins, our study may also shed light on regulations on target bindings by other long repeat-containing proteins. PMID:28841137

Investigation of the effect of mutations of rat albumin on the binding affinity to the alpha(4)beta(1) integrin antagonist, 4-[1-[3-chloro-4-[N'-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), using recombinant rat albumins.

PubMed

Ito, Takashi; Takahashi, Masayuki; Okazaki, Osamu; Sugiyama, Yuichi

2010-08-02

The authors reported previously rat strain differences in plasma protein binding to alpha(4)beta(1) antagonist D01-4582, resulting in a great strain difference in its pharmacokinetics (19-fold differences in the AUC). The previous study suggested that amino acid changes of V238L and/or T293I in albumin reduced the binding affinity. In order to elucidate the relative significance of these mutations, an expression system was developed to obtain recombinant rat albumins (rRSA) using Pichia pastoris, followed by a binding analysis of four rRSAs by the ultracentrifugation method. The equilibrium dissociation constant (K(d)) of wild-type rRSA was 210 nM, while K(d) of rRSA that carried both V238L and T293I mutations was 974 nM. K(d) of artificial rRSA that carried only V238L was 426 nM, and K(d) of artificial rRSA that carried only T293I was 191 nM. These results suggested that V238L would be more important in the alteration of K(d). However, since none of the single mutations were sufficient to explain the reduction of affinity, the possibility was also suggested that T293I interacted cooperatively to reduce the binding affinity of rat albumin to D01-4582. Further investigation is required to elucidate the mechanism of the possible cooperative interaction.

The Glycan Microarray Story from Construction to Applications.

PubMed

Hyun, Ji Young; Pai, Jaeyoung; Shin, Injae

2017-04-18

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

Profiling Charge Complementarity and Selectivity for Binding at the Protein Surface

PubMed Central

Sulea, Traian; Purisima, Enrico O.

2003-01-01

A novel analysis and representation of the protein surface in terms of electrostatic binding complementarity and selectivity is presented. The charge optimization methodology is applied in a probe-based approach that simulates the binding process to the target protein. The molecular surface is color coded according to calculated optimal charge or according to charge selectivity, i.e., the binding cost of deviating from the optimal charge. The optimal charge profile depends on both the protein shape and charge distribution whereas the charge selectivity profile depends only on protein shape. High selectivity is concentrated in well-shaped concave pockets, whereas solvent-exposed convex regions are not charge selective. This suggests the synergy of charge and shape selectivity hot spots toward molecular selection and recognition, as well as the asymmetry of charge selectivity at the binding interface of biomolecular systems. The charge complementarity and selectivity profiles map relevant electrostatic properties in a readily interpretable way and encode information that is quite different from that visualized in the standard electrostatic potential map of unbound proteins. PMID:12719221

Structural models of antibody variable fragments: A method for investigating binding mechanisms

NASA Astrophysics Data System (ADS)

Petit, Samuel; Brard, Frédéric; Coquerel, Gérard; Perez, Guy; Tron, François

1998-03-01

The value of comparative molecular modeling for elucidating structure-function relationships was demonstrated by analyzing six anti-nucleosome autoantibody variable fragments. Structural models were built using the automated procedure developed in the COMPOSER software, subsequently minimized with the AMBER force field, and validated according to several standard geometric and chemical criteria. Canonical class assignment from Chothia and Lesk's [Chottin and Lesk, J. Mol. Biol., 196 (1987) 901; Chothia et al., Nature, 342 (1989) 877] work was used as a supplementary validation tool for five of the six hypervariable loops. The analysis, based on the hypothesis that antigen binding could occur through electrostatic interactions, reveals a diversity of possible binding mechanisms of anti-nucleosome or anti-histone antibodies to their cognate antigen. These results lead us to postulate that anti-nucleosome autoantibodies could have different origins. Since both anti-DNA and anti-nculeosome autoantibodies are produced during the course of systemic lupus erythematosus, a non-organ specific autoimmune disease, a comparative structural and electrostatic analysis of the two populations of autoantibodies may constitute a way to elucidate their origin and the role of the antigen in tolerance breakdown. The present study illustrates some interests, advantages and limits of a methodology based on the use of comparative modeling and analysis of molecular surface properties.

Extensive variation at MHC DRB in the New Zealand sea lion (Phocarctos hookeri) provides evidence for balancing selection

PubMed Central

Osborne, A J; Zavodna, M; Chilvers, B L; Robertson, B C; Negro, S S; Kennedy, M A; Gemmell, N J

2013-01-01

Marine mammals are often reported to possess reduced variation of major histocompatibility complex (MHC) genes compared with their terrestrial counterparts. We evaluated diversity at two MHC class II B genes, DQB and DRB, in the New Zealand sea lion (Phocarctos hookeri, NZSL) a species that has suffered high mortality owing to bacterial epizootics, using Sanger sequencing and haplotype reconstruction, together with next-generation sequencing. Despite this species' prolonged history of small population size and highly restricted distribution, we demonstrate extensive diversity at MHC DRB with 26 alleles, whereas MHC DQB is dimorphic. We identify four DRB codons, predicted to be involved in antigen binding, that are evolving under adaptive evolution. Our data suggest diversity at DRB may be maintained by balancing selection, consistent with the role of this locus as an antigen-binding region and the species' recent history of mass mortality during a series of bacterial epizootics. Phylogenetic analyses of DQB and DRB sequences from pinnipeds and other carnivores revealed significant allelic diversity, but little phylogenetic depth or structure among pinniped alleles; thus, we could neither confirm nor refute the possibility of trans-species polymorphism in this group. The phylogenetic pattern observed however, suggests some significant evolutionary constraint on these loci in the recent past, with the pattern consistent with that expected following an epizootic event. These data may help further elucidate some of the genetic factors underlying the unusually high susceptibility to bacterial infection of the threatened NZSL, and help us to better understand the extent and pattern of MHC diversity in pinnipeds. PMID:23572124

Hydroxynitrile Lyases with α/β-Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

PubMed Central

Andexer, Jennifer N; Staunig, Nicole; Eggert, Thorsten; Kratky, Christoph; Pohl, Martina; Gruber, Karl

2012-01-01

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies. PMID:22851196

Stimulation of erythrocyte phosphatidylserine exposure by mercury ions

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

Eisele, Kerstin; Lang, Philipp A.; Kempe, Daniela S.

2006-01-15

The sequelae of mercury intoxication include induction of apoptosis. In nucleated cells, Hg{sup 2+}-induced apoptosis involves mitochondrial damage. The present study has been performed to elucidate effects of Hg{sup 2+} in erythrocytes which lack mitochondria but are able to undergo apoptosis-like alterations of the cell membrane. Previous studies have documented that activation of a Ca{sup 2+}-sensitive erythrocyte scramblase leads to exposure of phosphatidylserine at the erythrocyte surface, a typical feature of apoptotic cells. The erythrocyte scramblase is activated by osmotic shock, oxidative stress and/or energy depletion which increase cytosolic Ca{sup 2+} activity and/or activate a sphingomyelinase leading to formation ofmore » ceramide. Ceramide sensitizes the scramblase to Ca{sup 2+}. The present experiments explored the effect of Hg{sup 2+} ions on erythrocytes. Phosphatidylserine exposure after mercury treatment was estimated from annexin binding as determined in FACS analysis. Exposure to Hg{sup 2+} (1 {mu}M) indeed significantly increased annexin binding from 2.3 {+-} 0.5% (control condition) to 23 {+-} 6% (n = 6). This effect was paralleled by activation of a clotrimazole-sensitive K{sup +}-selective conductance as measured by patch-clamp recordings and by transient cell shrinkage. Further experiments revealed also an increase of ceramide formation by {approx}66% (n = 7) after challenge with mercury (1 {mu}M). In conclusion, mercury ions activate a clotrimazole-sensitive K{sup +}-selective conductance leading to transient cell shrinkage. Moreover, Hg{sup 2+} increases ceramide formation. The observed mechanisms could similarly participate in the triggering of apoptosis in nucleated cells by Hg{sup 2+}.« less

A selective EP4 PGE2 receptor agonist alleviates disease in a new mouse model of X-linked nephrogenic diabetes insipidus

PubMed Central

Li, Jian Hua; Chou, Chung-Lin; Li, Bo; Gavrilova, Oksana; Eisner, Christoph; Schnermann, Jürgen; Anderson, Stasia A.; Deng, Chu-Xia; Knepper, Mark A.; Wess, Jürgen

2009-01-01

X-linked nephrogenic diabetes insipidus (XNDI) is a severe kidney disease caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene that result in the loss of renal urine-concentrating ability. At present, no specific pharmacological therapy has been developed for XNDI, primarily due to the lack of suitable animal models. To develop what we believe to be the first viable animal model of XNDI, we generated mice in which the V2R gene could be conditionally deleted during adulthood by administration of 4-OH-tamoxifen. Radioligand-binding studies confirmed the lack of V2R-binding sites in kidneys following 4-OH-tamoxifen treatment, and further analysis indicated that upon V2R deletion, adult mice displayed all characteristic symptoms of XNDI, including polyuria, polydipsia, and resistance to the antidiuretic actions of vasopressin. Gene expression analysis suggested that activation of renal EP4 PGE2 receptors might compensate for the lack of renal V2R activity in XNDI mice. Strikingly, both acute and chronic treatment of the mutant mice with a selective EP4 receptor agonist greatly reduced all major manifestations of XNDI, including changes in renal morphology. These physiological improvements were most likely due to a direct action on EP4 receptors expressed on collecting duct cells. These findings illustrate the usefulness of the newly generated V2R mutant mice for elucidating and testing new strategies for the potential treatment of humans with XNDI. PMID:19729836

Ga3+ as a mechanistic probe in Fe3+ transport: characterization of Ga3+ interaction with FbpA.

PubMed

Weaver, Katherine D; Heymann, Jared J; Mehta, Arnav; Roulhac, Petra L; Anderson, Damon S; Nowalk, Andrew J; Adhikari, Pratima; Mietzner, Timothy A; Fitzgerald, Michael C; Crumbliss, Alvin L

2008-08-01

The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga(3+) and Fe(3+), focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga(3+) complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.

Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

PubMed Central

Wang, Weijun; Mai-Gisondi, Galina; Stogios, Peter J.; Kaur, Amrit; Xu, Xiaohui; Cui, Hong; Turunen, Ossi; Savchenko, Alexei

2014-01-01

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket. PMID:24951792

Global decrease of serotonin-1A receptor binding after electroconvulsive therapy in major depression measured by PET

PubMed Central

Lanzenberger, R; Baldinger, P; Hahn, A; Ungersboeck, J; Mitterhauser, M; Winkler, D; Micskei, Z; Stein, P; Karanikas, G; Wadsak, W; Kasper, S; Frey, R

2013-01-01

Electroconvulsive therapy (ECT) is a potent therapy in severe treatment-refractory depression. Although commonly applied in psychiatric clinical routine since decades, the exact neurobiological mechanism regarding its efficacy remains unclear. Results from preclinical and clinical studies emphasize a crucial involvement of the serotonin-1A receptor (5-HT1A) in the mode of action of antidepressant treatment. This includes associations between treatment response and changes in 5-HT1A function and density by antidepressants. Further, alterations of the 5-HT1A receptor are consistently reported in depression. To elucidate the effect of ECT on 5-HT1A receptor binding, 12 subjects with severe treatment-resistant major depression underwent three positron emission tomography (PET) measurements using the highly selective radioligand [carbonyl-11C]WAY100635, twice before (test–retest variability) and once after 10.08±2.35 ECT sessions. Ten patients (∼83%) were responders to ECT. The voxel-wise comparison of the 5-HT1A receptor binding (BPND) before and after ECT revealed a widespread reduction in cortical and subcortical regions (P<0.05 corrected), except for the occipital cortex and the cerebellum. Strongest reductions were found in regions consistently reported to be altered in major depression and involved in emotion regulation, such as the subgenual part of the anterior cingulate cortex (−27.5%), the orbitofrontal cortex (−30.1%), the amygdala (−31.8%), the hippocampus (−30.6%) and the insula (−28.9%). No significant change was found in the raphe nuclei. There was no significant difference in receptor binding in any region comparing the first two PET scans conducted before ECT. This PET study proposes a global involvement of the postsynaptic 5-HT1A receptor binding in the effect of ECT. PMID:22751491

The influence of combined magnesium and vanadate administration on the level of some elements in selected rat organs: V-Mg interactions and the role of iron-essential protein (DMT-1) in the mechanism underlying altered tissues iron level.

PubMed

Scibior, Agnieszka; Adamczyk, Agnieszka; Gołębiowska, Dorota; Niedźwiecka, Irmina; Fornal, Emilia

2014-04-01

The effect of 12 week co-administration of sodium metavanadate (SMV) and magnesium sulfate (MS) on the levels of some elements in selected rats' organs and an attempt to elucidate a role of divalent metal transporter 1 (DMT-1) in the mechanism(s) of the SMV-induced disorders in some tissue Fe homeostasis were studied. SMV taken up separately or in combination with MS may pose a risk of the rise and shortage of the total hepatic and splenic Fe and Cu contents, respectively, cerebral Fe deficiency, splenic Ca deposition, and the hepatic, renal, and cerebral DMT-1 down-regulation. When administered alone, SMV may also cause the decrease in the total renal Fe and Cu contents. A visible protective effect of Mg against the renal and cerebral V accumulation and the decrease in the renal Fe and Cu contents during the SMV-MS co-administration together with our previous findings suggest a beneficial role of Mg at SMV exposure. Further, the SMV-induced fall in total iron binding capacity (TIBC), reported previously, and its correlations with the hepatic, splenic, and cerebral Fe levels allow us to suggest that diminished TIBC could be partly involved in the mechanism(s) responsible for the dramatic redistribution of Fe in those tissues. Finally, DMT-1, which potentially could participate in the hepatic non-transferrin Fe-bound uptake, does not play a significant role in this process indicating the need for studying other Fe transporters to more precisely elucidate molecular mechanism(s) underlying the hepatic Fe loading in our experimental conditions.

Fluorescence studies on binding of pyrene and its derivatives to humic acid

NASA Astrophysics Data System (ADS)

Nakashima, K.; Maki, M.; Ishikawa, F.; Yoshikawa, T.; Gong, Y.-K.; Miyajima, T.

2007-07-01

Binding of pyrene (PyH) and its derivatives to humic acid (HA) has been studied by fluorescence spectroscopy. The nature of the interaction between HA and pyrene derivatives are extensively investigated by employing three derivatives ranging from anionic to cationic compounds: 1-pyrenebutylic acid (PyA), 1-pyrenemethanol (PyM), and 1-pyrenebutyltrimethylammonium bromide (PyB). Binding constants between HA and PyX (X = H, A, M, B) are obtained by steady-state fluorescence quenching techniques, and it is found that PyB has a markedly large binding constant among the pyrene family. This is attributed to a strong electrostatic interaction between cationic PyB and anionic HA. The result suggests that an electrostatic interaction plays a dominant role in binding of pyrenes to humic acid. The importance of electrostatic interaction was also confirmed by a salt effect on the binding constant. Influence of collisional quenching on the binding constant, which causes overestimation of the binding constant, was examined by time-resolved fluorescence spectroscopy as well as temperature effect in steady-state fluorescence measurements. It is elucidated that collisional quenching does not much bring overestimation into the binding constants.

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

USDA-ARS?s Scientific Manuscript database

ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of...

Homology modeling and in silico site directed mutagenesis of pyruvate ferredoxin oxidoreductase from Clostridium thermocellum.

PubMed

Saranyah, Kannuchamy; Kalva, Sukesh; Mukund, Nisha; Singh, Sanjeev Kumar; Saleena, Lilly M

2015-01-01

Pyruvate ferredoxin oxidoreductase is the crucial enzyme that involves in bioethanol synthesis pathway of Clostridium thermocellum. It is an ethanologenic organism but has been investigated less on its enzyme structure. The amino acid sequence of Pyruvate ferredoxin oxidoreductase was derived from UNIPROT and the screened crystal structure was taken as the template for homology modeling using MODELLER 9V11. The model was loop refined and was validated using RMSD, ProSA and PROCHECK. The docking and per residue interaction studies were carried out to elucidate the interaction energies of amino acid residues with pyruvate. To enhance the binding of pyruvate with the enzyme, mutation studies were carried out by replacing Thr31 as it had a less interaction energy. Out of 10 mutants, T31N, T31Q and T31G were selected using potential energy and the residual energy calculations. Five nanoseconds explicit MD simulations were run for apo, wild type and mutants T31N, T31Q and T31G using Desmond. RMSD, RMSF, distance plots and H-bonds analysis proved T31G to be a favorable mutant for binding of pyruvate. Thus, modeling PFOR would help in profound understanding of its structural clefts and mutation studies would aid in improving the enzyme efficiency.

Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes.

PubMed

Lang, Philipp A; Kaiser, Stephanie; Myssina, Swetlana; Birka, Christina; Weinstock, Christof; Northoff, Hinnak; Wieder, Thomas; Lang, Florian; Huber, Stephan M

2004-04-01

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.

Musashi Protein-directed Translational Activation of Target mRNAs Is Mediated by the Poly(A) Polymerase, Germ Line Development Defective-2*

PubMed Central

Cragle, Chad; MacNicol, Angus M.

2014-01-01

The mRNA-binding protein, Musashi, has been shown to regulate translation of select mRNAs and to control cellular identity in both stem cells and cancer cells. Within the mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed Musashi's capability to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly(A) polymerase germ line development defective-2 (GLD2) and map the association domain to 31 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation. PMID:24644291

Molecular modeling studies of novel retro-binding tripeptide active-site inhibitors of thrombin.

PubMed

Lau, W F; Tabernero, L; Sack, J S; Iwanowicz, E J

1995-08-01

A novel series of retro-binding tripeptide thrombin active-site inhibitors was recently developed (Iwanowicz, E. I. et al. J. Med. Chem. 1994, 37, 2111(1)). It was hypothesized that the binding mode for these inhibitors is similar to that of the first three N-terminal residues of hirudin. This binding hypothesis was subsequently verified when the crystal structure of a member of this series, BMS-183,507 (N-[N-[N-[4-(Aminoiminomethyl)amino[-1-oxobutyl]-L- phenylalanyl]-L-allo-threonyl]-L-phenylalanine, methyl ester), was determined (Taberno, L.J. Mol. Biol. 1995, 246, 14). The methodology for developing the binding models of these inhibitors, the structure-activity relationships (SAR) and modeling studies that led to the elucidation of the proposed binding mode is described. The crystal structure of BMS-183,507/human alpha-thrombin is compared with the crystal structure of hirudin/human alpha-thrombin (Rydel, T.J. et al. Science 1990, 249,227; Rydel, T.J. et al. J. Mol Biol. 1991, 221, 583; Grutter, M.G. et al. EMBO J. 1990, 9, 2361) and with the computational binding model of BMS-183,507.

The Structure of the ZMYND8/Drebrin Complex Suggests a Cytoplasmic Sequestering Mechanism of ZMYND8 by Drebrin.

PubMed

Yao, Ningning; Li, Jianchao; Liu, Haiyang; Wan, Jun; Liu, Wei; Zhang, Mingjie

2017-11-07

Malfunctions of the actin binding protein Drebrin have been implicated in various human diseases such as Alzheimer's disease, cognitive impairments, cancer, and digestive disorders, though with poorly understood mechanisms. The ADF-H domain of Drebrin does not contain actin binding and depolymerizing activity. Instead, it binds to a histone marker reader, ZMYND8. Here we present the high-resolution crystal structure of Drebrin ADF-H in complex with the ZMYND8 PHD-BROMO-PWWP tandem, elucidating the mechanistic basis governing the highly specific interaction of the two proteins. The structure reveals that the ZMYND8 PHD-BROMO-PWWP tandem forms a structural supramodule that is necessary for binding to Drebrin ADF-H. Drebrin ADF-H competes with modified histone for binding to ZMYND8. Binding of Drebrin can shuttle ZMYND8 from nucleus to cytoplasm in living cells. Taken together, our study uncovers a non-actin target binding mode for ADF-H domains, and suggests that Drebrin may regulate activities of epigenetic reader ZMYND8 via its cytoplasmic sequestration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Roles of Copper-Binding Proteins in Breast Cancer.

PubMed

Blockhuys, Stéphanie; Wittung-Stafshede, Pernilla

2017-04-20

Copper ions are needed in several steps of cancer progression. However, the underlying mechanisms, and involved copper-binding proteins, are mainly elusive. Since most copper ions in the body (in and outside cells) are protein-bound, it is important to investigate what copper-binding proteins participate and, for these, how they are loaded with copper by copper transport proteins. Mechanistic information for how some copper-binding proteins, such as extracellular lysyl oxidase (LOX), play roles in cancer have been elucidated but there is still much to learn from a biophysical molecular viewpoint. Here we provide a summary of copper-binding proteins and discuss ones reported to have roles in cancer. We specifically focus on how copper-binding proteins such as mediator of cell motility 1 (MEMO1), LOX, LOX-like proteins, and secreted protein acidic and rich in cysteine (SPARC) modulate breast cancer from molecular and clinical aspects. Because of the importance of copper for invasion/migration processes, which are key components of cancer metastasis, further insights into the actions of copper-binding proteins may provide new targets to combat cancer.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules.

PubMed

Newton, Ana S; Deiana, Luca; Puleo, David E; Cisneros, José A; Cutrona, Kara J; Schlessinger, Joseph; Jorgensen, William L

2017-06-08

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as K d results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules

PubMed Central

2017-01-01

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses. PMID:28626520

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase.

PubMed

Lee, Chi Chung; Fay, Aaron W; Weng, Tsu-Chien; Krest, Courtney M; Hedman, Britt; Hodgson, Keith O; Hu, Yilin; Ribbe, Markus W

2015-11-10

Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.

The effect of extracts of the roots of the stinging nettle (Urtica dioica) on the interaction of SHBG with its receptor on human prostatic membranes.

PubMed

Hryb, D J; Khan, M S; Romas, N A; Rosner, W

1995-02-01

Extracts from the roots of the stinging nettle (Urtica dioica) are used in the treatment of benign prostatic hyperplasia. The mechanisms underlying this treatment have not been elucidated. We set out to determine whether specific extracts from U. dioica had the ability to modulate the binding of sex hormone-binding globulin to its receptor on human prostatic membranes. Four substances contained in U. dioica were examined: an aqueous extract; an alcoholic extract; U. dioica agglutinin, and stigmasta-4-en-3-one. Of these, only the aqueous extract was active. It inhibited the binding of 125I-SHBG to its receptor. The inhibition was dose related, starting at about 0.6 mg/ml and completely inhibited binding at 10 mg/ml.

Crystallization and preliminary X-ray diffraction analysis of a high-affinity phosphate-binding protein endowed with phosphatase activity from Pseudomonas aeruginosa PAO1

PubMed Central

Djeghader, Ahmed; Gotthard, Guillaume; Suh, Andrew; Gonzalez, Daniel; Scott, Ken; Chabriere, Eric; Elias, Mikael

2013-01-01

In prokaryotes, phosphate starvation induces the expression of numerous phosphate-responsive genes, such as the pst operon including the high-affinity phosphate-binding protein (PBP or pstS) and alkaline phosphatases such as PhoA. This response increases the cellular inorganic phosphate import efficiency. Notably, some Pseudomonas species secrete, via a type-2 secretion system, a phosphate-binding protein dubbed LapA endowed with phosphatase activity. Here, the expression, purification, crystallization and X-ray data collection at 0.87 Å resolution of LapA are described. Combined with biochemical and enzymatic characterization, the structure of this intriguing phosphate-binding protein will help to elucidate the molecular origin of its phosphatase activity and to decipher its putative role in phosphate uptake. PMID:24100568

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

PubMed Central

Matts, Robert L.; Dixit, Anshuman; Peterson, Laura B.; Sun, Liang; Voruganti, Sudhakar; Kalyanaraman, Palgunan; Hartson, Steve D.; Verkhivker, Gennady M.; Blagg, Brian S. J.

2011-01-01

The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein. PMID:21548602

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

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

Deciphering the selectivity of Bombyx mori pheromone binding protein for bombykol over bombykal: a theoretical approach.

PubMed

Charlier, Landry; Antonczak, Serge; Jacquin-Joly, Emmanuelle; Cabrol-Bass, Daniel; Golebiowski, Jérôme

2008-12-22

In this article we report calculations dedicated to estimate the selectivity of the Bombyx mori pheromone binding protein towards the two closely related pheromonal components Bombykol and Bombykal. The selectivity is quantified by the binding free-energy difference, obtained either by the thermodynamic integration or by the MM-GBSA approach. In the latter, the selectivity is decomposed on a per-residue basis, which identifies the residues considered crucial for the selectivity of the protein for Bombykol over Bombykal. A discussion on the role of Bombyx mori pheromone binding protein is provided on the basis of these results.

Heat shock protein 70.1 (Hsp70.1) affects neuronal cell fate by regulating lysosomal acid sphingomyelinase.

PubMed

Zhu, Hong; Yoshimoto, Tanihiro; Yamashima, Tetsumori

2014-10-03

The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of chemical elements in the trophic levels of natural salt marshes.

PubMed

Kamiński, Piotr; Barczak, Tadeusz; Bennewicz, Janina; Jerzak, Leszek; Bogdzińska, Maria; Aleksandrowicz, Oleg; Koim-Puchowska, Beata; Szady-Grad, Małgorzata; Klawe, Jacek J; Woźniak, Alina

2016-06-01

The relationships between the bioaccumulation of Na, K, Ca, Mg, Fe, Zn, Cu, Mn, Co, Cd, and Pb, acidity (pH), salinity (Ec), and organic matter content within trophic levels (water-soil-plants-invertebrates) were studied in saline environments in Poland. Environments included sodium manufactures, wastes utilization areas, dumping grounds, and agriculture cultivation, where disturbed Ca, Mg, and Fe exist and the impact of Cd and Pb is high. We found Zn, Cu, Mn, Co, and Cd accumulation in the leaves of plants and in invertebrates. Our aim was to determine the selectivity exhibited by soil for nutrients and heavy metals and to estimate whether it is important in elucidating how these metals are available for plant/animal uptake in addition to their mobility and stability within soils. We examined four ecological plant groups: trees, shrubs, minor green plants, and water macrophytes. Among invertebrates, we sampled breastplates Malacostraca, small arachnids Arachnida, diplopods Diplopoda, small insects Insecta, and snails Gastropoda. A higher level of chemical elements was found in saline polluted areas (sodium manufactures and anthropogenic sites). Soil acidity and salinity determined the bioaccumulation of free radicals in the trophic levels measured. A pH decrease caused Zn and Cd to increase in sodium manufactures and an increase in Ca, Zn, Cu, Cd, and Pb in the anthropogenic sites. pH increase also caused Na, Mg, and Fe to increase in sodium manufactures and an increase in Na, Fe, Mn, and Co in the anthropogenic sites. There was a significant correlation between these chemical elements and Ec in soils. We found significant relationships between pH and Ec, which were positive in saline areas of sodium manufactures and negative in the anthropogenic and control sites. These dependencies testify that the measurement of the selectivity of cations and their fluctuation in soils provide essential information on the affinity and binding strength in these environments. The chemical elements accumulated in soils and plants; however, further flow is selective and variable. The selectivity exhibited by soil systems for nutrients and heavy metals is important in elucidating how these metals become available for plant/animal uptake and also their mobility and stability in soils.

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

The Synthetic Analogs of Oxygen-Binding Heme Proteins.

ERIC Educational Resources Information Center

Suslick, Kenneth S.; Reinert, Thomas J.

1985-01-01

Discusses model studies aimed at elucidating various ways in which molecular oxygen interacts with metalloproteins. The focus is on the chemistry of iron(II) porphyrins and their adducts with nitrogenous bases, carbon monoxide, and dioxygen, which are most relevant to the functional proteries of the heme proteins, hemoglobin, and myoglobin. (JN)

ION COMPOSITION ELUCIDATION (ICE)

EPA Science Inventory

Ion Composition Elucidation (ICE) utilizes selected ion recording with a double focusing mass spectrometer to simultaneously determine exact masses and relative isotopic abundances from mass peak profiles. These can be determined more accurately and at higher sensitivity ...

A Comparative Study of the Effectiveness of Two Strategies of Etymological Elaboration and Pictorial Elucidation on Idiom Learning: A Case of Young EFL Iranian Learners

ERIC Educational Resources Information Center

Haghshenas, Mahsa Sadat Mousavi; Hashemian, Mahmood

2016-01-01

This study examined the effect of etymological elaboration, pictorial elucidation, and integration of these 2 strategies on idiom learning by L2 learners. A total number of 80 homogeneous intermediate learners studying English at 3 language institutes in Isfahan, Iran, were selected. The intermediate participants were selected as the result of…

Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus

PubMed Central

Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

2012-01-01

Thioredoxin binding protein −2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein −2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein −2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein −2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein −2 in metabolic control. Enhancement of thioredoxin binding protein −2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein −2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein −2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β2-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus. PMID:22247597

Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR.

PubMed

Kim, Yea Woon; Kim, AeRi

2017-07-20

Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here we applied the CRISPR/spCas9 system to mutate the binding motifs of transcription factors. Binding motifs for erythroid specific transcription factors were mutated in the locus control region hypersensitive sites of the human β-globin locus. Guide RNAs targeting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying a human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs. These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors. ©2017 The Author(s).

Dynamic motif occupancy (DynaMO) analysis identifies transcription factors and their binding sites driving dynamic biological processes

PubMed Central

Kuang, Zheng; Ji, Zhicheng

2018-01-01

Abstract Biological processes are usually associated with genome-wide remodeling of transcription driven by transcription factors (TFs). Identifying key TFs and their spatiotemporal binding patterns are indispensable to understanding how dynamic processes are programmed. However, most methods are designed to predict TF binding sites only. We present a computational method, dynamic motif occupancy analysis (DynaMO), to infer important TFs and their spatiotemporal binding activities in dynamic biological processes using chromatin profiling data from multiple biological conditions such as time-course histone modification ChIP-seq data. In the first step, DynaMO predicts TF binding sites with a random forests approach. Next and uniquely, DynaMO infers dynamic TF binding activities at predicted binding sites using their local chromatin profiles from multiple biological conditions. Another landmark of DynaMO is to identify key TFs in a dynamic process using a clustering and enrichment analysis of dynamic TF binding patterns. Application of DynaMO to the yeast ultradian cycle, mouse circadian clock and human neural differentiation exhibits its accuracy and versatility. We anticipate DynaMO will be generally useful for elucidating transcriptional programs in dynamic processes. PMID:29325176

Evaluation of galectin binding by frontal affinity chromatography (FAC).

PubMed

Iwaki, Jun; Hirabayashi, Jun

2015-01-01

Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato1

PubMed Central

Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong

2015-01-01

Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening. PMID:25637453

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

PubMed

Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

2016-06-21

The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Identification of a misfolded region in superoxide dismutase 1 that is exposed in amyotrophic lateral sclerosis.

PubMed

Rotunno, Melissa S; Auclair, Jared R; Maniatis, Stephanie; Shaffer, Scott A; Agar, Jeffrey; Bosco, Daryl A

2014-10-10

Mutations and aberrant post-translational modifications within Cu,Zn-superoxide dismutase (SOD1) cause this otherwise protective enzyme to misfold, leading to amyotrophic lateral sclerosis (ALS). The C4F6 antibody selectively binds misfolded SOD1 in spinal cord tissues from postmortem human ALS cases, as well as from an ALS-SOD1 mouse model, suggesting that the C4F6 epitope reports on a pathogenic conformation that is common to misfolded SOD1 variants. To date, the residues and structural elements that comprise this epitope have not been elucidated. Using a chemical cross-linking and mass spectrometry approach, we identified the C4F6 epitope within several ALS-linked SOD1 variants, as well as an oxidized form of WT SOD1, supporting the notion that a similar misfolded conformation is shared among pathological SOD1 proteins. Exposure of the C4F6 epitope was modulated by the SOD1 electrostatic (loop VII) and zinc binding (loop IV) loops and correlated with SOD1-induced toxicity in a primary microglia activation assay. Site-directed mutagenesis revealed Asp(92) and Asp(96) as key residues within the C4F6 epitope required for the SOD1-C4F6 binding interaction. We propose that stabilizing the functional loops within SOD1 and/or obscuring the C4F6 epitope are viable therapeutic strategies for treating SOD1-mediated ALS. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of potential herbal inhibitor of acetylcholinesterase associated Alzheimer's disorders using molecular docking and molecular dynamics simulation.

PubMed

Seniya, Chandrabhan; Khan, Ghulam Jilani; Uchadia, Kuldeep

2014-01-01

Cholinesterase inhibitors (ChE-Is) are the standard for the therapy of AD associated disorders and are the only class of approved drugs by the Food and Drug Administration (FDA). Additionally, acetylcholinesterase (AChE) is the target for many Alzheimer's dementia drugs which block the function of AChE but have some side effects. Therefore, in this paper, an attempt was made to elucidate cholinesterase inhibition potential of secondary metabolite from Cannabis plant which has negligible or no side effect. Molecular docking of 500 herbal compounds, against AChE, was performed using Autodock 4.2 as per the standard protocols. Molecular dynamics simulations have also been carried out to check stability of binding complex in water for 1000 ps. Our molecular docking and simulation have predicted high binding affinity of secondary metabolite (C28H34N2O6) to AChE. Further, molecular dynamics simulations for 1000 ps suggest that ligand interaction with the residues Asp72, Tyr70-121-334, and Phe288 of AChE, all of which fall under active site/subsite or binding pocket, might be critical for the inhibitory activity of AChE. This approach might be helpful to understand the selectivity of the given drug molecule in the treatment of Alzheimer's disease. The study provides evidence for consideration of C28H34N2O6 as a valuable small ligand molecule in treatment and prevention of AD associated disorders and further in vitro and in vivo investigations may prove its therapeutic potential.

Identification of Potential Herbal Inhibitor of Acetylcholinesterase Associated Alzheimer's Disorders Using Molecular Docking and Molecular Dynamics Simulation

PubMed Central

Seniya, Chandrabhan; Khan, Ghulam Jilani; Uchadia, Kuldeep

2014-01-01

Cholinesterase inhibitors (ChE-Is) are the standard for the therapy of AD associated disorders and are the only class of approved drugs by the Food and Drug Administration (FDA). Additionally, acetylcholinesterase (AChE) is the target for many Alzheimer's dementia drugs which block the function of AChE but have some side effects. Therefore, in this paper, an attempt was made to elucidate cholinesterase inhibition potential of secondary metabolite from Cannabis plant which has negligible or no side effect. Molecular docking of 500 herbal compounds, against AChE, was performed using Autodock 4.2 as per the standard protocols. Molecular dynamics simulations have also been carried out to check stability of binding complex in water for 1000 ps. Our molecular docking and simulation have predicted high binding affinity of secondary metabolite (C28H34N2O6) to AChE. Further, molecular dynamics simulations for 1000 ps suggest that ligand interaction with the residues Asp72, Tyr70-121-334, and Phe288 of AChE, all of which fall under active site/subsite or binding pocket, might be critical for the inhibitory activity of AChE. This approach might be helpful to understand the selectivity of the given drug molecule in the treatment of Alzheimer's disease. The study provides evidence for consideration of C28H34N2O6 as a valuable small ligand molecule in treatment and prevention of AD associated disorders and further in vitro and in vivo investigations may prove its therapeutic potential. PMID:25054066

In vitro selection using a dual RNA library that allows primerless selection

PubMed Central

Jarosch, Florian; Buchner, Klaus; Klussmann, Sven

2006-01-01

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C. PMID:16855281

Elucidation of conformational states, dynamics, and mechanism of binding in human κ-opioid receptor complexes.

PubMed

Leonis, Georgios; Avramopoulos, Aggelos; Salmas, Ramin Ekhteiari; Durdagi, Serdar; Yurtsever, Mine; Papadopoulos, Manthos G

2014-08-25

Opioid G protein-coupled receptors (GPCRs) have been implicated in modulating pain, addiction, psychotomimesis, mood and memory, among other functions. We have employed the recently reported crystal structure of the human κ-opioid receptor (κ-OR) and performed molecular dynamics (MD), free energy, and ab initio calculations to elucidate the binding mechanism in complexes with antagonist JDTic and agonist SalA. The two systems were modeled in water and in DPPC lipid bilayers, in order to investigate the effect of the membrane upon conformational dynamics. MD and Atoms in Molecules (AIM) ab initio calculations for the complexes in water showed that each ligand was stabilized inside the binding site of the receptor through hydrogen bond interactions that involved residues Asp138 (with JDTic) and Gln115, His291, Leu212 (with SalA). The static description offered by the crystal structure was overcome to reveal a structural rearrangement of the binding pocket, which facilitated additional interactions between JDTic and Glu209/Tyr139. The role of Glu209 was emphasized, since it belongs to an extracellular loop that covers the binding site of the receptor and is crucial for ligand entrapment. The above interactions were retained in membrane complexes (SalA forms additional hydrogen bonds with Tyr139/312), except the Tyr139 interaction, which is abolished in the JDTic complex. For the first time, we report that JDTic alternates between a "V-shape" (stabilized via a water-mediated intramolecular interaction) and a more extended conformation, a feature that offers enough suppleness for effective binding. Moreover, MM-PBSA calculations showed that the more efficient JDTic binding to κ-OR compared to SalA (ΔGJDTic = -31.6 kcal mol(-1), ΔGSalA = -9.8 kcal mol(-1)) is attributed mostly to differences in electrostatic contributions. Importantly, our results are in qualitative agreement with the experiments (ΔGJDTic,exp = -14.4 kcal mol(-1), ΔGSalA,exp = -10.8 kcal mol(-1)). This study provides previously unattainable information on the dynamics of human κ-OR and insight on the rational design of drugs with improved pharmacological properties.

Conformational selection in protein binding and function

PubMed Central

Weikl, Thomas R; Paul, Fabian

2014-01-01

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on- and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single-molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction. PMID:25155241

Mediation by neurotensin-receptors of effects of neurotensin on self-stimulation of the medial prefrontal cortex.

PubMed Central

Fernández, R.; Sabater, R.; Sáez, J. A.; Montes, R.; Alba, F.; Ferrer, J. M.

1996-01-01

1 Intracortical microinjections of neurotensin (NT) selectively decreased intracranial self-stimulation (ICSS) of the medial prefrontal cortex in the rat. 2 To elucidate whether this effect is mediated by NT receptors or by the formation of NT-dopamine complexes, we investigated the effects on ICSS of intracortical microinjections of neurotensin (1-11), an NT fragment that forms extracellular complexes with dopamine but does not bind to NT receptors. 3 We also studied the effects of the peripheral administration of SR 48692, a selective antagonist of NT receptors, on the inhibition of ICSS produced by the intracortical administration of NT. 4 Unilateral microinjections of neurotensin (1-11) at doses of 10, 20 and 40 nmol into the medial prefrontal cortex did not change the basal ICSS rate of this area. 5 The intraperitoneal administration of SR 48692 at doses of 0.08 and 0.16 mg kg-1 30 min before microinjection of 10 nmol of NT into the medial prefrontal cortex, antagonized the inhibition of ICSS produced by the neuropeptide. 6 These results demonstrate that the inhibitory effect of NT on ICSS is mediated by NT receptors. PMID:8886412

Genomic variation in Plasmodium vivax malaria reveals regions under selective pressure

PubMed Central

Diez Benavente, Ernest; Ward, Zoe; Chan, Wilson; Mohareb, Fady R.; Sutherland, Colin J.; Roper, Cally; Campino, Susana

2017-01-01

Background Although Plasmodium vivax contributes to almost half of all malaria cases outside Africa, it has been relatively neglected compared to the more deadly P. falciparum. It is known that P. vivax populations possess high genetic diversity, differing geographically potentially due to different vector species, host genetics and environmental factors. Results We analysed the high-quality genomic data for 46 P. vivax isolates spanning 10 countries across 4 continents. Using population genetic methods we identified hotspots of selection pressure, including the previously reported MRP1 and DHPS genes, both putative drug resistance loci. Extra copies and deletions in the promoter region of another drug resistance candidate, MDR1 gene, and duplications in the Duffy binding protein gene (PvDBP) potentially involved in erythrocyte invasion, were also identified. For surveillance applications, continental-informative markers were found in putative drug resistance loci, and we show that organellar polymorphisms could classify P. vivax populations across continents and differentiate between Plasmodia spp. Conclusions This study has shown that genomic diversity that lies within and between P. vivax populations can be used to elucidate potential drug resistance and invasion mechanisms, as well as facilitate the molecular barcoding of the parasite for surveillance applications. PMID:28493919

Genomic variation in Plasmodium vivax malaria reveals regions under selective pressure.

PubMed

Diez Benavente, Ernest; Ward, Zoe; Chan, Wilson; Mohareb, Fady R; Sutherland, Colin J; Roper, Cally; Campino, Susana; Clark, Taane G

2017-01-01

Although Plasmodium vivax contributes to almost half of all malaria cases outside Africa, it has been relatively neglected compared to the more deadly P. falciparum. It is known that P. vivax populations possess high genetic diversity, differing geographically potentially due to different vector species, host genetics and environmental factors. We analysed the high-quality genomic data for 46 P. vivax isolates spanning 10 countries across 4 continents. Using population genetic methods we identified hotspots of selection pressure, including the previously reported MRP1 and DHPS genes, both putative drug resistance loci. Extra copies and deletions in the promoter region of another drug resistance candidate, MDR1 gene, and duplications in the Duffy binding protein gene (PvDBP) potentially involved in erythrocyte invasion, were also identified. For surveillance applications, continental-informative markers were found in putative drug resistance loci, and we show that organellar polymorphisms could classify P. vivax populations across continents and differentiate between Plasmodia spp. This study has shown that genomic diversity that lies within and between P. vivax populations can be used to elucidate potential drug resistance and invasion mechanisms, as well as facilitate the molecular barcoding of the parasite for surveillance applications.

STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism

NASA Astrophysics Data System (ADS)

Létourneau, Danny; Bédard, Mikaël; Cabana, Jérôme; Lefebvre, Andrée; Lehoux, Jean-Guy; Lavigne, Pierre

2016-06-01

START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.

Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods.

PubMed

Zhang, Guowen; Ma, Yadi

2013-01-15

The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA. Copyright © 2012 Elsevier Ltd. All rights reserved.

An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide-Calcium Complex.

PubMed

Sun, Na; Jin, Ziqi; Li, Dongmei; Yin, Hongjie; Lin, Songyi

2017-11-08

The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates and calcium ions was elucidated upon its structural and thermodynamics characteristics. The present study demonstrated that the DHTKE peptide could spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex on improving the calcium absorption was investigated in vitro using Caco-2 cells. Results showed that the DHTKE-calcium complex could facilitate the calcium influx into the cytosol and further improve calcium absorption across Caco-2 cell monolayers by more than 7 times when compared to calcium-free control. This study facilitates the understanding about the binding mechanism between peptides and calcium ions as well as suggests a potential application of egg white peptides as nutraceuticals to improve calcium absorption.

An RNA-Binding Multimer Specifies Nematode Sperm Fate.

PubMed

Aoki, Scott T; Porter, Douglas F; Prasad, Aman; Wickens, Marvin; Bingman, Craig A; Kimble, Judith

2018-06-26

FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Collective Dynamics of Periplasmic Glutamine Binding Protein upon Domain Closure

PubMed Central

Loeffler, Hannes H.; Kitao, Akio

2009-01-01

The glutamine binding protein is a vital component of the associated ATP binding cassette transport systems responsible for the uptake of glutamine into the cell. We have investigated the global movements of this protein by molecular dynamics simulations and principal component analysis (PCA). We confirm that the most dominant mode corresponds to the biological function of the protein, i.e., a hinge-type motion upon ligand binding. The closure itself was directly observed from two independent trajectories whereby PCA was used to elucidate the nature of this closing reaction. Two intermediary states are identified and described in detail. The ligand binding induces the structural change of the hinge regions from a discontinuous β-sheet to a continuous one, which also enhances softness of the hinge and modifies the direction of hinge motion to enable closing. We also investigated the convergence behavior of PCA modes, which were found to converge rather quickly when the associated magnitudes of the eigenvalues are well separated. PMID:19883597

Selection of the simplest RNA that binds isoleucine

PubMed Central

LOZUPONE, CATHERINE; CHANGAYIL, SHANKAR; MAJERFELD, IRENE; YARUS, MICHAEL

2003-01-01

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA–amino acid binding structures. PMID:14561881

The yeast transcription elongation factor Spt4/5 is a sequence‐specific RNA binding protein

PubMed Central

Blythe, Amanda J.; Yazar‐Klosinski, Berra; Webster, Michael W.; Chen, Eefei; Vandevenne, Marylène; Bendak, Katerina; Mackay, Joel P.; Hartzog, Grant A.

2016-01-01

Abstract The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co‐transcriptional pre‐mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence‐specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA‐binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5. PMID:27376968

Mechanism of lignin inhibition of enzymatic biomass deconstruction

DOE PAGES

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong; ...

2015-12-01

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

Insights into finding a mismatch through the structure of a mispaired DNA bound by a rhodium intercalator

PubMed Central

Pierre, Valérie C.; Kaiser, Jens T.; Barton, Jacqueline K.

2007-01-01

We report the 1.1-Å resolution crystal structure of a bulky rhodium complex bound to two different DNA sites, mismatched and matched in the oligonucleotide 5′-(dCGGAAATTCCCG)2-3′. At the AC mismatch site, the structure reveals ligand insertion from the minor groove with ejection of both mismatched bases and elucidates how destabilized mispairs in DNA may be recognized. This unique binding mode contrasts with major groove intercalation, observed at a matched site, where doubling of the base pair rise accommodates stacking of the intercalator. Mass spectral analysis reveals different photocleavage products associated with the two binding modes in the crystal, with only products characteristic of mismatch binding in solution. This structure, illustrating two clearly distinct binding modes for a molecule with DNA, provides a rationale for the interrogation and detection of mismatches. PMID:17194756

Identification of Human Lineage-Specific Transcriptional Coregulators Enabled by a Glossary of Binding Modules and Tunable Genomic Backgrounds.

PubMed

Mariani, Luca; Weinand, Kathryn; Vedenko, Anastasia; Barrera, Luis A; Bulyk, Martha L

2017-09-27

Transcription factors (TFs) control cellular processes by binding specific DNA motifs to modulate gene expression. Motif enrichment analysis of regulatory regions can identify direct and indirect TF binding sites. Here, we created a glossary of 108 non-redundant TF-8mer "modules" of shared specificity for 671 metazoan TFs from publicly available and new universal protein binding microarray data. Analysis of 239 ENCODE TF chromatin immunoprecipitation sequencing datasets and associated RNA sequencing profiles suggest the 8mer modules are more precise than position weight matrices in identifying indirect binding motifs and their associated tethering TFs. We also developed GENRE (genomically equivalent negative regions), a tunable tool for construction of matched genomic background sequences for analysis of regulatory regions. GENRE outperformed four state-of-the-art approaches to background sequence construction. We used our TF-8mer glossary and GENRE in the analysis of the indirect binding motifs for the co-occurrence of tethering factors, suggesting novel TF-TF interactions. We anticipate that these tools will aid in elucidating tissue-specific gene-regulatory programs. Copyright © 2017 Elsevier Inc. All rights reserved.

Binding interactions of halogenated bisphenol A with mouse PPARα: In vitro investigation and molecular dynamics simulation.

PubMed

Zhang, Jie; Li, Tiezhu; Wang, Tuoyi; Guan, Tianzhu; Yu, Hansong; Li, Zhuolin; Wang, Yongzhi; Wang, Yongjun; Zhang, Tiehua

2018-02-01

The binding of bisphenol A (BPA) and its halogenated derivatives (halogenated BPAs) to mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD) was examined by a combination of in vitro investigation and in silico simulation. Fluorescence polarization (FP) assay showed that halogenated BPAs could bind to mPPARα-LBD* as the affinity ligands. The calculated electrostatic potential (ESP) illustrated the different charge distributions of halogenated BPAs with altered halogenation patterns. As electron-attracting substituents, halogens decrease the positive electrostatic potential and thereby have a significant influence on the electrostatic interactions of halogenated BPAs with mPPARα-LBD*. The docking results elucidated that hydrophobic and hydrogen-bonding interactions may also contribute to stabilize the binding of the halogenated BPAs to their receptor molecule. Comparison of the calculated binding energies with the experimentally determined affinities yielded a good correlation (R 2 =0.6659) that could provide a rational basis for designing environmentally benign chemicals with reduced toxicities. This work can potentially be used for preliminary screening of halogenated BPAs. Copyright © 2017 Elsevier B.V. All rights reserved.

Methyl group reorientation under ligand binding probed by pseudocontact shifts.

PubMed

Lescanne, Mathilde; Ahuja, Puneet; Blok, Anneloes; Timmer, Monika; Akerud, Tomas; Ubbink, Marcellus

2018-06-02

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1-3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.

The 1.3 A resolution structure of the RNA tridecamer r(GCGUUUGAAACGC): metal ion binding correlates with base unstacking and groove contraction.

PubMed

Timsit, Youri; Bombard, Sophie

2007-12-01

Metal ions play a key role in RNA folding and activity. Elucidating the rules that govern the binding of metal ions is therefore an essential step for better understanding the RNA functions. High-resolution data are a prerequisite for a detailed structural analysis of ion binding on RNA and, in particular, the observation of monovalent cations. Here, the high-resolution crystal structures of the tridecamer duplex r(GCGUUUGAAACGC) crystallized under different conditions provides new structural insights on ion binding on GAAA/UUU sequences that exhibit both unusual structural and functional properties in RNA. The present study extends the repertory of RNA ion binding sites in showing that the two first bases of UUU triplets constitute a specific site for sodium ions. A striking asymmetric pattern of metal ion binding in the two equivalent halves of the palindromic sequence demonstrates that sequence and its environment act together to bind metal ions. A highly ionophilic half that binds six metal ions allows, for the first time, the observation of a disodium cluster in RNA. The comparison of the equivalent halves of the duplex provides experimental evidences that ion binding correlates with structural alterations and groove contraction.

Essential role of conformational selection in ligand binding.

PubMed

Vogt, Austin D; Pozzi, Nicola; Chen, Zhiwei; Di Cera, Enrico

2014-02-01

Two competing and mutually exclusive mechanisms of ligand recognition - conformational selection and induced fit - have dominated our interpretation of ligand binding in biological macromolecules for almost six decades. Conformational selection posits the pre-existence of multiple conformations of the macromolecule from which the ligand selects the optimal one. Induced fit, on the other hand, postulates the existence of conformational rearrangements of the original conformation into an optimal one that are induced by binding of the ligand. In the former case, conformational transitions precede the binding event; in the latter, conformational changes follow the binding step. Kineticists have used a facile criterion to distinguish between the two mechanisms based on the dependence of the rate of relaxation to equilibrium, kobs, on the ligand concentration, [L]. A value of kobs decreasing hyperbolically with [L] has been seen as diagnostic of conformational selection, while a value of kobs increasing hyperbolically with [L] has been considered diagnostic of induced fit. However, this simple conclusion is only valid under the rather unrealistic assumption of conformational transitions being much slower than binding and dissociation events. In general, induced fit only produces values of kobs that increase with [L] but conformational selection is more versatile and is associated with values of kobs that increase with, decrease with or are independent of [L]. The richer repertoire of kinetic properties of conformational selection applies to kinetic mechanisms with single or multiple saturable relaxations and explains the behavior of nearly all experimental systems reported in the literature thus far. Conformational selection is always sufficient and often necessary to account for the relaxation kinetics of ligand binding to a biological macromolecule and is therefore an essential component of any binding mechanism. On the other hand, induced fit is never necessary and only sufficient in a few cases. Therefore, the long assumed importance and preponderance of induced fit as a mechanism of ligand binding should be reconsidered. © 2013 Elsevier B.V. All rights reserved.

Elucidation of the glucose transport pathway in glucose transporter 4 via steered molecular dynamics simulations.

PubMed

Sheena, Aswathy; Mohan, Suma S; Haridas, Nidhina Pachakkil A; Anilkumar, Gopalakrishnapillai

2011-01-01

GLUT4 is a predominant insulin regulated glucose transporter expressed in major glucose disposal tissues such as adipocytes and muscles. Under the unstimulated state, GLUT4 resides within intracellular vesicles. Various stimuli such as insulin translocate this protein to the plasma membrane for glucose transport. In the absence of a crystal structure for GLUT4, very little is known about the mechanism of glucose transport by this protein. Earlier we proposed a homology model for GLUT4 and performed a conventional molecular dynamics study revealing the conformational rearrangements during glucose and ATP binding. However, this study could not explain the transport of glucose through the permeation tunnel. To elucidate the molecular mechanism of glucose transport and its energetic, a steered molecular dynamics study (SMD) was used. Glucose was pulled from the extracellular end of GLUT4 to the cytoplasm along the pathway using constant velocity pulling method. We identified several key residues within the tunnel that interact directly with either the backbone ring or the hydroxyl groups of glucose. A rotation of glucose molecule was seen near the sugar binding site facilitating the sugar recognition process at the QLS binding site. This study proposes a possible glucose transport pathway and aids the identification of several residues that make direct interactions with glucose during glucose transport. Mutational studies are required to further validate the observation made in this study.

Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

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

Boone, Christopher D.; Tu, Chingkuang; McKenna, Robert, E-mail: rmckenna@ufl.edu

The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes tomore » mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.« less

Binding Pathway of Opiates to μ-Opioid Receptors Revealed by Machine Learning

NASA Astrophysics Data System (ADS)

Barati Farimani, Amir; Feinberg, Evan; Pande, Vijay

2018-02-01

Many important analgesics relieve pain by binding to the $\\mu$-Opioid Receptor ($\\mu$OR), which makes the $\\mu$OR among the most clinically relevant proteins of the G Protein Coupled Receptor (GPCR) family. Despite previous studies on the activation pathways of the GPCRs, the mechanism of opiate binding and the selectivity of $\\mu$OR are largely unknown. We performed extensive molecular dynamics (MD) simulation and analysis to find the selective allosteric binding sites of the $\\mu$OR and the path opiates take to bind to the orthosteric site. In this study, we predicted that the allosteric site is responsible for the attraction and selection of opiates. Using Markov state models and machine learning, we traced the pathway of opiates in binding to the orthosteric site, the main binding pocket. Our results have important implications in designing novel analgesics.

Molecular dynamic simulations reveal the structural determinants of fatty acid binding to oxy-myoglobin

USDA-ARS?s Scientific Manuscript database

The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolite...

Discovery of Tertiary Sulfonamides as Potent Liver X Receptor Antagonists

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

Zuercher, William J.; Buckholz†, Richard G.; Campobasso, Nino

2010-08-12

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

PubMed

Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

2010-04-22

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation

NASA Astrophysics Data System (ADS)

Li, Yi-Tong; Yi, Cong; Chen, Chen-Chen; Lan, Huan; Pan, Man; Zhang, Shao-Jin; Huang, Yi-Chao; Guan, Chao-Jian; Li, Yi-Ming; Yu, Li; Liu, Lei

2017-03-01

Acetylation of Atg3 regulates the lipidation of the protein Atg8 in autophagy. The molecular mechanism behind this important biochemical event remains to be elucidated. We describe the first semi-synthesis of homogeneous K19/K48-diacetylated Atg3 through sequential hydrazide-based native chemical ligation. In vitro reconstitution experiments with the semi-synthetic proteins confirm that Atg3 acetylation can promote the lipidation of Atg8. We find that acetylation of Atg3 enhances its binding to phosphatidylethanolamine-containing liposomes and to endoplasmic reticulum, through which it promotes the lipidation process.

Understanding chemical binding using the Berlin function and the reaction force

NASA Astrophysics Data System (ADS)

Chakraborty, Debajit; Cárdenas, Carlos; Echegaray, Eleonora; Toro-Labbe, Alejandro; Ayers, Paul W.

2012-06-01

We use the derivative of the electron density with respect to the reaction coordinate, interpreted through the Berlin binding function, to identify portions of the reaction path where chemical bonds are breaking and forming. The results agree with the conventional description for SN2 reactions, but they are much more general and can be used to elucidate other types of reactions also. Our analysis offers support for, and detailed information about, the use of the reaction force profile to separate the reaction coordinates into intervals, each with characteristic extents of geometry change and electronic rearrangement.

Predicting Selectivity of Uranium vs. Vanadium from First Principles: Complete Molecular Design and Adsorption Modeling

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

Ivanov, Aleksandr; Das, Sadananda; Bryantsev, Vyacheslav

SUMMARYBackground: Uranium is used as the basic fuel for nuclear power plants, which generate significant amounts of electricity and have life cycle carbon emissions that are as low as renewable energy sources. The extraction of this valuable energy commodity from the ground remains controversial, however, mainly because of environmental and health impacts. Alternatively, seawater offers an enormous uranium resource that may be tapped at minimal environmental cost. Currently, amidoxime polymers are the most widely considered adsorbent materials for large-scale extraction of uranium from seawater, but they are not perfectly selective for uranyl, UO22+. In particular, the competition between UO22+ andmore » vanadium (VO2+/VO2+) cations poses a significant challenge to the efficient mining of UO22+. Thus, accelerating progress in the discovery and deployment of advanced materials for the recovery of uranium relies on the design of new ligands with high binding affinity and selectivity for uranium over competing metal ions. A cost-effective route to aid the discovery of new ligands is to apply computational methods to rapidly test attractive candidates and elucidate data-driven guidelines for rational design.Objectives: One of the key components in achieving rational design of highly selective ligands is the establishment of computational tools capable of assessing ligand selectivity trends. Therefore, the objectives of this study include:1.Establish first-principles methods, based on computational chemistry techniques, to calculate stability constants for UO22+ and VO2+/VO2+ complexes.2.Develop computational protocols to assess the binding strengths and selectivity of ligands that can be present in the actual poly(acrylamidoxime) adsorbents.3.Develop adsorption models that can use information from first-principles computational methods to predict the adsorption behavior of uranium and vanadium by adsorbents synthesized at ORNL and compare results with experimental data.Results: In the first part of the study, we present an approach based on quantum chemical calculations that achieves high accuracy in reproducing experimental aqueous stability constants for UO22+ and VO2+/VO2+ complexes, providing the essential foundation for prospective screening of existing or even yet unsynthesized ligands with higher selectivity for uranium over vanadium. The developed computational protocols were used to assess the binding strengths and selectivity of aliphatic dicarboxylate ligands that can be present in the actual poly(acrylamidoxime) adsorbents. It was found that simple dicarboxylic functional groups possess low binding affinity and selectivity for uranyl, because their backbones present architectures that are poorly organized for the UO22+ complexation. In the second part of the study, adsorption models were developed and coupled with the results of the molecular studies in an effort to predict the adsorption of uranium and vanadium by the ORNL AF1 fiber adsorbent. These models can account for the effects of ligand surface density, specific surface area, surface charging, and other non-idealities of the adsorbent surface. It was found that by utilizing the reaction schemes and binding strengths proposed by the molecular studies, the adsorption model could accurately predict the uptake of uranium by both the acyclic amidoxime (AO) and cyclic imide dioximate (IDO) ligand in the ORNL laboratory studies, especially in the more neutral pH ranges (pH 5 to 9). The model, however, performed less adequately for predicting the uptake of vanadium for the same adsorbent. By exploring the causes behind the discrepancy between the adsorption model and the ORNL laboratory data, we found that the effect of surface charging was suppressing the total vanadium uptake predicted by the model. An investigation of literature revealed that the crystal structures of the 1:2 vanadium/IDO complex involved a sodium ion (Na+), which neutralized the charge of the adsorbed species. When this charge neutral species was included in the adsorption model, the predictions of the vanadium capacities were significantly improved across all pH ranges. This result suggests that, during adsorption, some surface species may closely associate with, or adsorb, counter-ions from solution to neutralize the charge of the adsorbent surface.Significance: This study is particularly significant when considering whether to produce an otherwise highly attractive ligand that may be synthetically challenging. If such a ligand is predicted by our calculations to achieve the desired uranium vs vanadium selectivity, this substantially reduces the risk of taking on such synthetic challenges. Moreover, the elimination of ligands that are unlikely to show a good uranyl binding affinity can release resources to focus on more promising UO22+- selective ligands. Furthermore, the results can successfully rationalize the experimentally observed loss in selectivity of amidoxime-based fibers« less

Computational design of nanoparticle drug delivery systems for selective targeting

NASA Astrophysics Data System (ADS)

Duncan, Gregg A.; Bevan, Michael A.

2015-09-01

Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues.Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues. Electronic supplementary information (ESI) available: Movie showing simulation renderings of targeted (ρL = 1820/μm2, KD = 120 μM) nanoparticle selective binding to cancer (ρR = 256/μm2) vs. healthy (ρR = 64/μm2) cell surfaces. Target membrane proteins have linear color scale depending on binding energy ranging from white when unbound (URL = 0) to red when tightly bound (URL = UM). See DOI: 10.1039/c5nr03691g

Is there a link between selectivity and binding thermodynamics profiles?

PubMed

Tarcsay, Ákos; Keserű, György M

2015-01-01

Thermodynamics of ligand binding is influenced by the interplay between enthalpy and entropy contributions of the binding event. The impact of these binding free energy components, however, is not limited to the primary target only. Here, we investigate the relationship between binding thermodynamics and selectivity profiles by combining publicly available data from broad off-target assay profiling and the corresponding thermodynamics measurements. Our analysis indicates that compounds binding their primary targets with higher entropy contributions tend to hit more off-targets compared with those ligands that demonstrated enthalpy-driven binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Spectroscopic and molecular modeling approaches to investigate the interaction of bisphenol A, bisphenol F and their diglycidyl ethers with PPARα.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Ruan, Ping; Ren, Dayong; Dai, Weichang; Yu, Hansong; Li, Tiezhu

2017-08-01

A fluorescence polarization (FP) assay for the simultaneous determination of bisphenol A (BPA), bisphenol F (BPF), bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) was developed. The method was based on the competition between bisphenols (BPs) and fluorescein-labeled dexamethasone derivative (Dex-fl) for mouse peroxisome proliferator-activated receptor α ligand binding domain (mPPARα-LBD). A recombinant soluble protein derivative mPPARα-LBD* was prepared, then in vitro binding of 4 BPs to mPPARα-LBD* was investigated. Fluorescence polarization assay showed that these compounds exhibited different binding potencies with mPPARα-LBD*. Additionally, molecular dynamics simulations were performed to further understand the mechanism of BPs binding affinity for mPPARα-LBD*. Docking results elucidated that the driving forces for the binding of BPs to mPPARα-LBD* were predominantly dependent on hydrophobic and hydrogen-bonding interactions. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.7258). The proposed method has potential for multi-residue detection of BPA, BPF, BADGE, and BFDGE. Copyright © 2017 Elsevier Ltd. All rights reserved.

Elucidating the mechanism of action of pregabalin: α(2)δ as a therapeutic target in anxiety.

PubMed

Micó, Juan-Antonio; Prieto, Rita

2012-08-01

This review provides a brief summary of what is known about the anxiolytic mechanism of action of pregabalin, a highly selective, high-affinity ligand of the P/Q type of voltage-gated calcium channel (CaV). Evidence from in vivo models of neuronal hyperexcitability suggests that pregabalin reduces synaptic release of neurotransmitters in selected CNS regions including the cortex, olfactory bulb, hypothalamus, amygdala, hippocampus, cerebellum and dorsal horn of the spinal cord. Release of neurotransmitters from the synaptic vesicle, and propagation of neurotransmission, requires the vesicle to fuse with the presynaptic membrane. Pregabalin binding to the α(2)δ type 1 protein of the P/Q type CaV reduces the availability of Ca2+ required for membrane fusion and exocytosis of neurotransmitters. Evidence that the anxiolytic mechanism of action of pregabalin is mediated by binding to the α(2)δ type 1 protein comes from animal models, which have demonstrated a structure-activity relationship between the affinity of ligands for the α(2)δ type 1 protein and their potency in models of anxiety such as the Vogel conflict test. Furthermore, the anxiolytic activity of pregabalin is lost in transgenic mice with specific point mutations in the CaV α(2)δ type 1 protein. Pregabalin-mediated reduction in calcium currents has also been shown to result in a significant inhibition of the release of neurotransmitters implicated in pathological anxiety such as glutamate and monoamine neurotransmitters. However, further research is needed to confirm that these effects contribute to the anxiolytic mechanism of action of pregabalin. Finally, pregabalin may also act by inhibiting synaptogenesis of excitatory neurons formed in response to chronic stress or anxiety, or more acutely inhibit the trafficking of CaV to the plasma membrane.

Presynaptic selectivity of a ligand for serotonin 1A receptors revealed by in vivo PET assays of rat brain.

PubMed

Saijo, Takeaki; Maeda, Jun; Okauchi, Takashi; Maeda, Jun-ichi; Morio, Yasunori; Kuwahara, Yasuhiro; Suzuki, Masayuki; Goto, Nobuharu; Fukumura, Toshimitsu; Suhara, Tetsuya; Higuchi, Makoto

2012-01-01

A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT(1A)) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT(1A) receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT(1A) receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT(1A) receptors. In addition, [(35)S]guanosine 5'-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT(1A) receptors. This finding has lent support to reports that diverse partial agonists for 5-HT(1A) receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants.

Presynaptic Selectivity of a Ligand for Serotonin 1A Receptors Revealed by In Vivo PET Assays of Rat Brain

PubMed Central

Okauchi, Takashi; Maeda, Jun-ichi; Morio, Yasunori; Kuwahara, Yasuhiro; Suzuki, Masayuki; Goto, Nobuharu; Fukumura, Toshimitsu; Suhara, Tetsuya; Higuchi, Makoto

2012-01-01

A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT1A) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT1A receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT1A receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT1A receptors. In addition, [35S]guanosine 5′-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT1A receptors. This finding has lent support to reports that diverse partial agonists for 5-HT1A receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants. PMID:22880045

Insights into the complex formed by matrix metalloproteinase-2 and alloxan inhibitors: molecular dynamics simulations and free energy calculations.

PubMed

Giangreco, Ilenia; Lattanzi, Gianluca; Nicolotti, Orazio; Catto, Marco; Laghezza, Antonio; Leonetti, Francesco; Stefanachi, Angela; Carotti, Angelo

2011-01-01

Matrix metalloproteinases (MMP) are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. Hence, the development of potent and selective inhibitors targeting these enzymes continues to be eagerly sought. In this paper, a number of alloxan-based compounds, initially conceived to bias other therapeutically relevant enzymes, were rationally modified and successfully repurposed to inhibit MMP-2 (also named gelatinase A) in the nanomolar range. Importantly, the alloxan core makes its debut as zinc binding group since it ensures a stable tetrahedral coordination of the catalytic zinc ion in concert with the three histidines of the HExxHxxGxxH metzincin signature motif, further stabilized by a hydrogen bond with the glutamate residue belonging to the same motif. The molecular decoration of the alloxan core with a biphenyl privileged structure allowed to sample the deep S(1)' specificity pocket of MMP-2 and to relate the high affinity towards this enzyme with the chance of forming a hydrogen bond network with the backbone of Leu116 and Asn147 and the side chains of Tyr144, Thr145 and Arg149 at the bottom of the pocket. The effect of even slight structural changes in determining the interaction at the S(1)' subsite of MMP-2 as well as the nature and strength of the binding is elucidated via molecular dynamics simulations and free energy calculations. Among the herein presented compounds, the highest affinity (pIC(50) = 7.06) is found for BAM, a compound exhibiting also selectivity (>20) towards MMP-2, as compared to MMP-9, the other member of the gelatinases.

Shape recognition of microbial cells by colloidal cell imprints

NASA Astrophysics Data System (ADS)

Borovička, Josef; Stoyanov, Simeon D.; Paunov, Vesselin N.

2013-08-01

We have engineered a class of colloids which can recognize the shape and size of targeted microbial cells and selectively bind to their surfaces. These imprinted colloid particles, which we called ``colloid antibodies'', were fabricated by partial fragmentation of silica shells obtained by templating the targeted microbial cells. We successfully demonstrated the shape and size recognition between such colloidal imprints and matching microbial cells. High percentage of binding events of colloidal imprints with the size matching target particles was achieved. We demonstrated selective binding of colloidal imprints to target microbial cells in a binary mixture of cells of different shapes and sizes, which also resulted in high binding selectivity. We explored the role of the electrostatic interactions between the target cells and their colloid imprints by pre-coating both of them with polyelectrolytes. Selective binding occurred predominantly in the case of opposite surface charges of the colloid cell imprint and the targeted cells. The mechanism of the recognition is based on the amplification of the surface adhesion in the case of shape and size match due to the increased contact area between the target cell and the colloidal imprint. We also tested the selective binding for colloid imprints of particles of fixed shape and varying sizes. The concept of cell recognition by colloid imprints could be used for development of colloid antibodies for shape-selective binding of microbes. Such colloid antibodies could be additionally functionalized with surface groups to enhance their binding efficiency to cells of specific shape and deliver a drug payload directly to their surface or allow them to be manipulated using external fields. They could benefit the pharmaceutical industry in developing selective antimicrobial therapies and formulations.

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box.

PubMed

Jia, Chuandong; Zuo, Wei; Yang, Dong; Chen, Yanming; Cao, Liping; Custelcean, Radu; Hostaš, Jiří; Hobza, Pavel; Glaser, Robert; Wang, Yao-Yu; Yang, Xiao-Juan; Wu, Biao

2017-10-16

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. This similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives, as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, L-carnitine, and glycine betaine effectively.The choline-binding protein ChoX exhibits a synergistic dual-site binding mode that allows it to discriminate choline over structural analogues. Here, the authors design a biomimetic triple anion helicate receptor whose selectivity for choline arises from a similar binding mechanism.

The Unique α4(+)/(−)α4 Agonist Binding Site in (α4)3(β2)2 Subtype Nicotinic Acetylcholine Receptors Permits Differential Agonist Desensitization Pharmacology versus the (α4)2(β2)3 Subtype

PubMed Central

Eaton, J. Brek; Lucero, Linda M.; Stratton, Harrison; Chang, Yongchang; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.

2014-01-01

Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using 86Rb+ efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(−)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit–specific [125I]mAb295 labeling. However, maximum function is approximately five times greater on a “per-receptor” basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(−)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(−)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development. PMID:24190916

Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases the expression of prostaglandin E2 receptor subtype EP4. The roles of phosphatidylinositol 3-kinase and CCAAT/enhancer-binding protein beta.

PubMed

Han, ShouWei; Ritzenthaler, Jeffrey D; Wingerd, Byron; Roman, Jesse

2005-09-30

The prostaglandin E2 receptor subtype EP4 has been implicated in the growth and progression of human non-small cell lung carcinoma (NSCLC). However, the factors that control its expression have not been entirely elucidated. Our studies show that NSCLC cells express peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) protein and that treatment with a selective PPARbeta/delta agonist (GW501516) increases EP4 mRNA and protein levels. GW501516 induced NSCLC cell proliferation, and this effect was prevented by PPARbeta/delta antisense or EP4 short interfering RNA (siRNA). GW501516 increased the phosphorylation of Akt and decreased PTEN expression. The selective inhibitor of phosphatidylinositol 3-kinase (PI3-K), wortmannin, and PPARbeta/delta antisense, abrogated the effect of GW501516 on EP4 expression, whereas that of the inhibitor of Erk did not. GW501516 also increased EP4 promoter activity through effects on the region between -1555 and -992 bp in the EP4 promoter, and mutation of the CCAAT/enhancer-binding protein (C/EBP) site in this region abrogated the effect of GW501516. GW501516 increased not only the binding activity of C/EBP to the NF-IL6 site in the EP4 promoter, which was prevented by the inhibitor of PI3-K, but also increased C/EBPbeta protein in a dose- and PPARbeta/delta-dependent manner. The effect of GW501516 on EP4 protein was eliminated in the presence of C/EBPbeta siRNA. Finally, we showed that pretreatment of NSCLC with GW501516 further increased NSCLC cell proliferation in response to exogenous dimethyl-prostaglandin E2 (PGE2) that was diminished in the presence of PPARbeta/delta antisense and EP4 siRNA. Taken together, these findings suggest that activation of PPARbeta/delta induces PGE2 receptor subtype EP4 expression through PI3-K signals and increases human lung carcinoma cell proliferation in response to PGE2. The increase in transcription of the EP4 gene by PPARbeta/delta agonist was associated with increased C/EBP binding activity in the NF-IL6 site of EP4 promoter region and C/EBPbeta protein expression that were mediated through both PI3-K/Akt and PPARbeta/delta signaling pathways.

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

PubMed Central

Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S.; Højrup, Peter; Poulsen, Steen S.; Nexo, Ebba

2012-01-01

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins. PMID:22872637

Network analysis reveals the recognition mechanism for complex formation of mannose-binding lectins

NASA Astrophysics Data System (ADS)

Jian, Yiren; Zhao, Yunjie; Zeng, Chen

The specific carbohydrate binding of lectin makes the protein a powerful molecular tool for various applications including cancer cell detection due to its glycoprotein profile on the cell surface. Most biologically active lectins are dimeric. To understand the structure-function relation of lectin complex, it is essential to elucidate the short- and long-range driving forces behind the dimer formation. Here we report our molecular dynamics simulations and associated dynamical network analysis on a particular lectin, i.e., the mannose-binding lectin from garlic. Our results, further supported by sequence coevolution analysis, shed light on how different parts of the complex communicate with each other. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.

Synthesis and binding affinity of neuropeptide Y at opiate receptors.

PubMed

Kiddle, James J; McCreery, Heather J; Soles, Sonia

2003-03-24

Neuropeptide Y and several metabolic fragments were synthesized and evaluated for binding affinity at non-selective opiate receptors. Neuropeptide Y and several C-terminal fragments were shown to bind to non-selective opiate receptors with an affinity similar to that of Leu-enkephalin.

Discriminating binding mechanisms of an intrinsically disordered protein via a multi-state coarse-grained model

NASA Astrophysics Data System (ADS)

Knott, Michael; Best, Robert B.

2014-05-01

Many proteins undergo a conformational transition upon binding to their cognate binding partner, with intrinsically disordered proteins (IDPs) providing an extreme example in which a folding transition occurs. However, it is often not clear whether this occurs via an "induced fit" or "conformational selection" mechanism, or via some intermediate scenario. In the first case, transient encounters with the binding partner favour transitions to the bound structure before the two proteins dissociate, while in the second the bound structure must be selected from a subset of unbound structures which are in the correct state for binding, because transient encounters of the incorrect conformation with the binding partner are most likely to result in dissociation. A particularly interesting situation involves those intrinsically disordered proteins which can bind to different binding partners in different conformations. We have devised a multi-state coarse-grained simulation model which is able to capture the binding of IDPs in alternate conformations, and by applying it to the binding of nuclear coactivator binding domain (NCBD) to either ACTR or IRF-3 we are able to determine the binding mechanism. By all measures, the binding of NCBD to either binding partner appears to occur via an induced fit mechanism. Nonetheless, we also show how a scenario closer to conformational selection could arise by choosing an alternative non-binding structure for NCBD.

Lipid A binding proteins in macrophages detected by ligand blotting

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

Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

1987-05-01

Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessedmore » using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.« less

Dynamic motif occupancy (DynaMO) analysis identifies transcription factors and their binding sites driving dynamic biological processes.

PubMed

Kuang, Zheng; Ji, Zhicheng; Boeke, Jef D; Ji, Hongkai

2018-01-09

Biological processes are usually associated with genome-wide remodeling of transcription driven by transcription factors (TFs). Identifying key TFs and their spatiotemporal binding patterns are indispensable to understanding how dynamic processes are programmed. However, most methods are designed to predict TF binding sites only. We present a computational method, dynamic motif occupancy analysis (DynaMO), to infer important TFs and their spatiotemporal binding activities in dynamic biological processes using chromatin profiling data from multiple biological conditions such as time-course histone modification ChIP-seq data. In the first step, DynaMO predicts TF binding sites with a random forests approach. Next and uniquely, DynaMO infers dynamic TF binding activities at predicted binding sites using their local chromatin profiles from multiple biological conditions. Another landmark of DynaMO is to identify key TFs in a dynamic process using a clustering and enrichment analysis of dynamic TF binding patterns. Application of DynaMO to the yeast ultradian cycle, mouse circadian clock and human neural differentiation exhibits its accuracy and versatility. We anticipate DynaMO will be generally useful for elucidating transcriptional programs in dynamic processes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

PubMed

Takakusagi, Yoichi; Kuramochi, Kouji; Takagi, Manami; Kusayanagi, Tomoe; Manita, Daisuke; Ozawa, Hiroko; Iwakiri, Kanako; Takakusagi, Kaori; Miyano, Yuka; Nakazaki, Atsuo; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo

2008-11-15

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

PubMed

Sosic, Alice; Saccone, Irene; Carraro, Caterina; Kenderdine, Thomas; Gamba, Elia; Caliendo, Giuseppe; Corvino, Angela; Di Vaio, Paola; Fiorino, Ferdinando; Magli, Elisa; Perissutti, Elisa; Santagada, Vincenzo; Severino, Beatrice; Spada, Valentina; Fabris, Dan; Frecentese, Francesco; Gatto, Barbara

2018-06-12

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

Structural characterization of human galectin-4 C-terminal domain: elucidating the molecular basis for recognition of glycosphingolipids, sulfated saccharides and blood group antigens.

PubMed

Bum-Erdene, Khuchtumur; Leffler, Hakon; Nilsson, Ulf J; Blanchard, Helen

2015-09-01

Human galectin-4 is a lectin that is expressed mainly in the gastrointestinal tract and exhibits metastasis-promoting roles in some cancers. Its tandem-repeat nature exhibits two distinct carbohydrate recognition domains allowing crosslinking by simultaneous binding to sulfated and non-sulfated (but not sialylated) glycosphingolipids and glycoproteins, facilitating stabilization of lipid rafts. Critically, galectin-4 exerts favourable or unfavourable effects depending upon the cancer. Here we report the first X-ray crystallographic structural information on human galectin-4, specifically the C-terminal carbohydrate recognition domain of human (galectin-4C) in complex with lactose, lactose-3'-sulfate, 2'-fucosyllactose, lacto-N-tetraose and lacto-N-neotetraose. These structures enable elucidation of galectin-4C binding fine-specificity towards sulfated and non-sulfated lacto- and neolacto-series sphingolipids as well as to human blood group antigens. Analysis of the lactose-3'-sulfate complex structure shows that galectin-4C does not recognize the sulfate group using any specific amino acid, but binds the ligand nonetheless. Complex structures with lacto-N-tetraose and lacto-N-neotetraose displayed differences in binding interactions exhibited by the non-reducing-end galactose. That of lacto-N-tetraose points outward from the protein surface whereas that of lacto-N-neotetraose interacts directly with the protein. Recognition patterns of human galectin-4C towards lacto- and neolacto-series glycosphingolipids are similar to those of human galectin-3; however, detailed scrutiny revealed differences stemming from the extended binding site that offer distinction in ligand profiles of these two galectins. Structural characterization of the complex with 2'-fucosyllactose, a carbohydrate with similarity to the H antigen, and molecular dynamics studies highlight structural features that allow specific recognition of A and B antigens, whilst a lack of interaction with the 2'-fucose of blood group antigens was revealed. 4YLZ, 4YM0, 4YM1, 4YM2, 4YM3. © 2015 FEBS.

Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

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

Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroupmore » (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.« less

Inhibition of the EGFR/STAT3/CEBPD Axis Reverses Cisplatin Cross-resistance with Paclitaxel in the Urothelial Carcinoma of the Urinary Bladder.

PubMed

Wang, Wei-Jan; Li, Chien-Feng; Chu, Yu-Yi; Wang, Yu-Hui; Hour, Tzyh-Chyuan; Yen, Chia-Jui; Chang, Wen-Chang; Wang, Ju-Ming

2017-01-15

Cisplatin (CDDP) is frequently used in combination chemotherapy with paclitaxel for treating urothelial carcinoma of the urinary bladder (UCUB). CDDP cross-resistance has been suggested to develop with paclitaxel, thus hindering successful UCUB treatment. Therefore, elucidating the mechanisms underlying CDDP-induced anticancer drug resistance is imperative and may provide an insight in developing novel therapeutic strategy. Loss-of-function assays were performed to elucidate the role of the EGFR and STAT3 in CDDP-induced CCAAT/enhancer-binding protein delta (CEBPD) expression in UCUB cells. Reporter and in vivo DNA-binding assays were employed to determine whether CEBPD directly regulates ATP binding cassette subfamily B member 1 (ABCB1) and ATP binding cassette subfamily C member 2 (ABCC2) activation. Finally, a xenograft animal assay was used to examine the abilities of gefitinib and S3I-201 (a STAT3 inhibitor) to reverse CDDP and paclitaxel sensitivity. CEBPD expression was maintained in postoperative chemotherapy patients, and this expression was induced by CDDP even in CDDP-resistant UCUB cells. Upon CDDP treatment, CEBPD activated ABCB1 and ABCC2. Furthermore, the EGFR/STAT3 pathway contributed to CDDP-induced CEBPD expression in UCUB cells. Gefitinib and S3I-201 treatment significantly reduced the expression of CEBPD and enhanced the sensitivity of CDDP-resistant UCUB cells to CDDP and paclitaxel. Our results revealed the risk of CEBPD activation in CDDP-resistant UCUB cells and suggested a therapeutic strategy for patients with UCUB or UCUB resisted to CDDP and paclitaxel by combination with either gefitinib or S3I-201. Clin Cancer Res; 23(2); 503-13. ©2016 AACR. ©2016 American Association for Cancer Research.

Molecular basis for the Kallmann syndrome-linked fibroblast growth factor receptor mutation

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

Thurman, Ryan D.; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy

Highlights: Black-Right-Pointing-Pointer The structural basis of the Kallmann syndrome is elucidated. Black-Right-Pointing-Pointer Kallmann syndrome mutation (A168S) induces a subtle conformational change(s). Black-Right-Pointing-Pointer Structural interactions mediated by beta-sheet G are most perturbed. Black-Right-Pointing-Pointer Ligand (FGF)-receptor interaction(s) is completely abolished by Kallmann mutation. Black-Right-Pointing-Pointer Kallmann mutation directly affects the FGF signaling process. -- Abstract: Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associatedmore » KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. {sup 1}H-{sup 15}N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR.« less

Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines.

PubMed

Latypov, Vitaly F; Tubbs, Julie L; Watson, Amanda J; Marriott, Andrew S; McGown, Gail; Thorncroft, Mary; Wilkinson, Oliver J; Senthong, Pattama; Butt, Amna; Arvai, Andrew S; Millington, Christopher L; Povey, Andrew C; Williams, David M; Santibanez-Koref, Mauro F; Tainer, John A; Margison, Geoffrey P

2012-07-13

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural and Dynamic Insights into the Mechanism of Allosteric Signal Transmission in ERK2-Mediated MKP3 Activation.

PubMed

Lu, Chang; Liu, Xin; Zhang, Chen-Song; Gong, Haipeng; Wu, Jia-Wei; Wang, Zhi-Xin

2017-11-21

The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the 334 FNFM 337 motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and is responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the time scale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2-induced MKP3 activation.

Hepatic manifestations of women with polycystic ovary syndrome.

PubMed

Chen, Mei-Jou; Ho, Hong-Nerng

2016-11-01

Women with polycystic ovary syndrome (PCOS) have a higher prevalence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) than the general population. The link between NAFLD/NASH and PCOS is not just a coincidence. Indeed, both of these disorders comprise common risk factors, including central obesity, insulin resistance, chronic low-grade inflammation, and hyperandrogenemia. The characteristics of hyperandrogenemia in women with PCOS include elevated total and free testosterone levels and low sex hormone-binding globulin levels and are reported to be associated with NAFLD and elevated liver enzymes; however, not all elevated androgen levels in women with PCOS have the same adverse effects on the liver. With the exception of weight loss and encouraging exercise in obese women, few evidence-based effective treatments target NAFLD/NASH in women with PCOS. Selective antiandrogens and insulin sensitizers might be beneficial in treating NAFLD/NASH in women with PCOS, but further elucidation is needed. Copyright © 2016. Published by Elsevier Ltd.

The antibiotic CJ-15,801 is an antimetabolite that hijacks and then inhibits CoA biosynthesis.

PubMed

van der Westhuyzen, Renier; Hammons, Justin C; Meier, Jordan L; Dahesh, Samira; Moolman, Wessel J A; Pelly, Stephen C; Nizet, Victor; Burkart, Michael D; Strauss, Erick

2012-05-25

The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics and highlight CoA biosynthesis as a viable antimicrobial drug target. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis and profiling of a 3-aminopyridin-2-one-based kinase targeted fragment library: Identification of 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one scaffold for monopolar spindle 1 (MPS1) and Aurora kinases inhibition.

PubMed

Fearon, Daren; Westwood, Isaac M; van Montfort, Rob L M; Bayliss, Richard; Jones, Keith; Bavetsias, Vassilios

2018-07-15

Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Elucidating the influence of polymorph-dependent interfacial solvent structuring at chitin surfaces.

PubMed

Brown, Aaron H; Walsh, Tiffany R

2016-10-20

Interfacial solvent structuring is thought to be influential in mediating the adsorption of biomolecules at aqueous materials interfaces. However, despite the enormous potential for exploitation of aqueous chitin interfaces in industrial, medical and drug-delivery applications, little is known at the molecular-level about such interfacial solvent structuring for chitin. Here we use molecular simulation to predict the structure of the [100] and [010] interfaces of α-chitin and β-chitin dihydrate in contact with liquid water and saline solution. We find the α-chitin [100] interface supports lateral high-density regions in the first water layer at the interface, which are also present, but not as pronounced, for β-chitin. The lateral structuring of interfacial ions at the saline/chitin interface is also more pronounced for α-chitin compared with β-chitin. Our findings provide a foundation for the systematic design of biomolecules with selective binding affinity for different chitin polymorphs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Clenbuterol Induces Cell Cycle Arrest in C2C12 Myoblasts by Delaying p27 Degradation through β-arrestin 2 Signaling

PubMed Central

Chen, Min; Liu, Chuncheng; Wang, Meng; Wang, Hong; Zhang, Kuo; Zheng, Yu; Yu, Zhengquan; Li, Xiangdong; Guo, Wei; Li, Ning; Meng, Qingyong

2017-01-01

β2-Adrenoceptor (β2-AR) agonists promote muscle growth. The aim of this study was to elucidate some effects of the selective β2-adrenoceptor agonist clenbuterol (CLB) on myoblast proliferation. We found that CLB induces cell cycle arrest in C2C12 myoblasts. This effect is partly due to the enhanced stability of p27, rather than the increased gene transcription via cAMP response element-binding protein (CREB). Specifically, CLB treatment enhanced the accumulation of p27 in the nucleus while depleting it from the cytosol via a mechanism that requires β2-AR. Surprisingly, p27 accumulation was not reversed by the protein kinase A (PKA) inhibitor H-89, but interestingly, was alleviated by the knockdown of β-arrestin 2. Thus, our work provides a basis for β2-AR agonists inhibit myoblasts proliferation through signaling via β2-AR, β-arrestin 2, and p27. PMID:29104500

Evolution of AF6-RAS association and its implications in mixed-lineage leukemia

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

Smith, Matthew J.; Ottoni, Elizabeth; Ishiyama, Noboru

Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding ofmore » MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.« less

Chemistry of carcinogenic metals.

PubMed Central

Martell, A E

1981-01-01

The periodic distribution of known and suspected carcinogenic metal ions is described, and the chemical behavior of various types of metal ions is explained in terms of the general theory of hard and soft acids and bases. The chelate effect is elucidated, and the relatively high stability of metal chelates in very dilute solutions is discussed. The concepts employed for the chelate effect are extended to explain the high stabilities of macrocyclic and cryptate complexes. Procedures for the use of equilibrium data to determine the speciation of metal ions and complexes under varying solution conditions are described. Methods for assessing the interferences by hydrogen ion, competing metal ions, hydrolysis, and precipitation are explained, and are applied to systems containing iron(III) chelates of fourteen chelating agents designed for effective binding of the ferric ion. The donor groups available for the building up of multidentate ligands are presented, and the ways in which they may be combined to achieve high affinity and selectivity for certain types of metal ions are explained. PMID:6791915

Methotrexate in Atherogenesis and Cholesterol Metabolism

PubMed Central

Coomes, Eric; Chan, Edwin S. L.; Reiss, Allison B.

2011-01-01

Methotrexate is a disease-modifying antirheumatic drug commonly used to treat inflammatory conditions such as rheumatoid arthritis which itself is linked to increased cardiovascular risk. Treatments that target inflammation may also impact the cardiovascular system. While methotrexate improves cardiovascular risk, inhibition of the cyclooxygenase (COX)-2 enzyme promotes atherosclerosis. These opposing cardiovascular influences may arise from differing effects on the expression of proteins involved in cholesterol homeostasis. These proteins, ATP-binding cassette transporter (ABC) A1 and cholesterol 27-hydroxylase, facilitate cellular cholesterol efflux and defend against cholesterol overload. Methotrexate upregulates expression of cholesterol 27-hydroxylase and ABCA1 via adenosine release, while COX-2 inhibition downregulates these proteins. Adenosine, acting through the A2A and A3 receptors, may upregulate proteins involved in reverse cholesterol transport by cAMP-PKA-CREB activation and STAT inhibition, respectively. Elucidating underlying cardiovascular mechanisms of these drugs provides a framework for developing novel cardioprotective anti-inflammatory medications, such as selective A2A receptor agonists. PMID:21490773

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

PubMed Central

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

PubMed

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

Control of Ion Selectivity in LeuT: Two Na+ Binding Sites with two different mechanisms

PubMed Central

Noskov, Sergei Y.; Roux, Benoît

2016-01-01

The x-ray structure of LeuT, a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporter, provides a great opportunity to better understand the molecular basis of monovalent cation selectivity in ion-coupled transporters. LeuT possesses two ion-binding sites, NA1 and NA2, which are highly selective for Na+. Extensive all-atom free energy molecular dynamics simulations of LeuT embedded in an explicit membrane are performed at different temperatures and various occupancy states of the binding sites to dissect the molecular mechanism of ion selectivity. The results show that the two binding sites display robust selectivity for Na+ over K+ or Li+, the competing ions of most similar radii. Of particular interest, the mechanism primarily responsible for selectivity for each of the two binding sites appears to be different. In site NA1, selectivity for Na+ over K+ arises predominantly from the strong electrostatic field arising from the negatively charged carboxylate group of the leucine substrate coordinating the ion directly. In site NA2, which comprises only neutral ligands, selectivity for Na+ is enforced by the local structural restraints arising from the hydrogen-bonding network and the covalent connectivity of the poly-peptide chain surrounding the ion according to a snug-fit mechanism. PMID:18280500

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes[W

PubMed Central

Rühl, Christina; Stauffer, Eva; Kahles, André; Wagner, Gabriele; Drechsel, Gabriele; Rätsch, Gunnar; Wachter, Andreas

2012-01-01

Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5′ splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid–dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner. PMID:23192226

C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia

PubMed Central

Yamasaki, Sho; Matsumoto, Makoto; Takeuchi, Osamu; Matsuzawa, Tetsuhiro; Ishikawa, Eri; Sakuma, Machie; Tateno, Hiroaki; Uno, Jun; Hirabayashi, Jun; Mikami, Yuzuru; Takeda, Kiyoshi; Akira, Shizuo; Saito, Takashi

2009-01-01

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRγ-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRγ, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to α-mannose but not mannan. Thus, Mincle may recognize specific geometry of α-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle−/− mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle−/− mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus. PMID:19171887

A critical review of fundamental controversies in the field of GPR30 research.

PubMed

Langer, Gernot; Bader, Benjamin; Meoli, Luca; Isensee, Jörg; Delbeck, Martina; Noppinger, Patricia Ruiz; Otto, Christiane

2010-01-01

The female sex hormone estradiol plays an important role in reproduction, mammary gland development, bone turnover, metabolism, and cardiovascular function. The effects of estradiol are mediated by two classical nuclear receptors, estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). In 2005, G-protein-coupled receptor 30 (GPR30) was claimed to act as a non-classical estrogen receptor that was also activated by the ERalpha and ERbeta antagonists tamoxifen and fulvestrant (ICI 182780). Despite many conflicting results regarding the potential role of GPR30 as an estrogen receptor, the official nomenclature was changed to GPER (G-protein-coupled estrogen receptor). This review revisits the inconsistencies that still exist in the literature and focuses on selected publications that basically address the following two questions: what is the evidence for and against the hypothesis that GPR30 acts as an estrogen receptor? What is the potential in vivo role of GPR30? Thus, in the first part we focus on conflicting results from in vitro studies analysing the subcellular localization of GPR30, its ability to bind (or not to bind) estradiol and to signal (or not to signal) in response to estradiol. In the second part, we discuss the strengths and limitations of four available GPR30 mouse models. We elucidate the potential impact of different targeting strategies on phenotypic diversity. Copyright 2010 Elsevier Inc. All rights reserved.

Antidiuretic effects of a novel nonpeptide vasopressin V(2)-receptor agonist, OPC-51803, administered orally to dogs.

PubMed

Nakamura, Shigeki; Hirano, Takahiro; Onogawa, Toshiyuki; Itoh, Shuji; Hashimoto, Ayako; Yamamura, Yoshitaka; Kondo, Kazumi; Mori, Toyoki; Kambe, Toshimi

2004-04-01

We elucidated the pharmacological properties of a novel nonpeptide vasopressin V(2)-receptor agonist, OPC-51803 ((5R)-2-[1-(2-chloro-4-(1-pyrrolidinyl)benzoyl-2,3,4,5-tetrahydro-1H-1-benzazepine-5-yl]-N-isopropylacetamide), via both in vitro binding experiments incorporating canine kidney and platelet membrane fractions and in vivo experiments that would determine the compound's antidiuretic effects after oral administration to water-loaded dogs. OPC-51803 displaced [(3)H]arginine vasopressin (AVP) binding to canine V(2) and V(1a) receptors, as determined by resulting K(i) values of 15.2 +/- 0.6 nM (n = 4) and 653 +/- 146 nM (n = 4), respectively. These data indicate that OPC-51803 was about 43 times more selective for V(2) receptors than for V(1a) receptors. Antidiuretic studies showed that orally administered doses of OPC-51803 (0.03 to 0.3 mg x kg(-1)) decreased urine volume and increased urinary osmolality in a dose-dependent manner in water-loaded dogs. Intravenous OPC-51803 infusions (0.3 and 3 microg x kg(-1) x min(-1)) did not affect renal or systemic hemodynamics in anesthetized dogs. Since these results confirm that OPC-51803 shows antidiuretic action in dogs, the compound may be useful for treating AVP-deficient pathophysiological states.

Efficient Capture of Perrhenate and Pertechnetate by a Mesoporous Zr Metal–Organic Framework and Examination of Anion Binding Motifs

DOE PAGES

Drout, Riki J.; Otake, Kenichi; Howarth, Ashlee J.; ...

2018-01-10

At the Hanford Site in southeastern Washington state, the U.S. Department of Energy intends to treat 56 million gallons of legacy nuclear waste by encasing it in borosilicate glass via vitrification. This process ineffectively captures radioactive pertechnetate (TcO 4–) because of the ion’s volatility, thereby requiring a different remediation method for this long-lived (t 1/2 = 2.1 × 10 5 years), environmentally mobile species. Currently available sorbents lack the desired combination of high uptake capacity, fast kinetics, and selectivity. Here, we evaluate the ability of the chemically and thermally robust Zr 6-based metal–organic framework (MOF), NU-1000, to capture perrhenate (ReOmore » 4–), a pertechnetate simulant, and pertechnetate. Our material exhibits an excellent perrhenate uptake capacity of 210 mg/g, reaches saturation within 5 min, and maintains perrhenate uptake in the presence of competing anions. Additionally, experiments with pertechnetate confirm perrhenate is a suitable surrogate. Single-crystal X-ray diffraction indicates both chelating and nonchelating perrhenate binding motifs are present in both the small pore and the mesopore of NU-1000. Postadsorption diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) further elucidates the uptake mechanism and powder X-ray diffraction (PXRD) and Brunauer–Emmett–Teller (BET) surface area analysis confirm the retention of crystallinity and porosity of NU-1000 throughout adsorption.« less

A vicious loop of fatty acid-binding protein 4 and DNA methyltransferase 1 promotes acute myeloid leukemia and acts as a therapeutic target

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhao, N; Zhang, YW; Bode, AM; Al-Kali, A; Litzow, MR; Li, B; Liu, SJ

2017-01-01

Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMT) is a key hallmark of acute myeloid leukemia (AML), yet efforts to target DNMT deregulation for drug development have lagged. We previously demonstrated that upregulation of fatty acid-binding protein 4 (FABP4) promotes AML aggressiveness through enhanced DNMT1-dependent DNA methylation. Here we demonstrate that FABP4 upregulation in AML cells occurs through vascular endothelial growth factor (VEGF) signaling, thus elucidating a crucial FABP4-DNMT1 regulatory feedback loop in AML biology. We show that FABP4 dysfunction by its selective inhibitor BMS309403 leads to downregulation of DNMT1, decrease of global DNA methylation and re-expression of p15INK4B tumor suppressor gene by promoter DNA hypomethylation in vitro, ex vivo and in vivo. Functionally, BMS309403 suppresses cell colony formation, induces cell differentiation, and, importantly, impairs leukemic disease progression in mouse models of leukemia. Our findings highlight AML-promoting properties of the FABP4-DNMT1 vicious loop, and identify an attractive class of therapeutic agents with a high potential for clinical use in AML patients. The results will also assist in establishing the FABP4-DNMT1 loop as a target for therapeutic discovery to enhance the index of current epigenetic therapies. PMID:28993705

Integration of Nuclear- and Extranuclear-Initiated Estrogen Receptor Signaling in Breast Cancer Cells

ERIC Educational Resources Information Center

Madak Erdogan, Zeynep

2009-01-01

Estrogenic hormones exert their effects through binding to Estrogen Receptors (ERs), which work in concert with coregulators and extranuclear signaling pathways to control gene expression in normal as well as cancerous states, including breast tumors. In this thesis, we have used multiple genome-wide analysis tools to elucidate various ways that…

ENZYMATIC COUPLING OF THE HERBICIDE BENTAZON WITH HUMUS MONOMERS AND CHARACTERIZATION OF REACTION PRODUCTS (R823847)

EPA Science Inventory

To elucidate the binding mechanism of the herbicide bentazon
(3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide) with
humic monomers in the presence of an oxidative enzyme, the reaction of bentazon
with catechol, caffeic acid, protocatechuic...

Tilting the balance between canonical and noncanonical conformations for the H1 hypervariable loop of a llama VHH through point mutations.

PubMed

Mahajan, Sai Pooja; Velez-Vega, Camilo; Escobedo, Fernando A

2013-01-10

Nanobodies are single-domain antibodies found in camelids. These are the smallest naturally occurring binding domains and derive functionality via three hypervariable loops (H1-H3) that form the binding surface. They are excellent candidates for antibody engineering because of their favorable characteristics like small size, high solubility, and stability. To rationally engineer antibodies with affinity for a specific target, the hypervariable loops can be tailored to obtain the desired binding surface. As a first step toward such a goal, we consider the design of loops with a desired conformation. In this study, we focus on the H1 loop of the anti-hCG llama nanobody that exhibits a noncanonical conformation. We aim to "tilt" the stability of the H1 loop structure from a noncanonical conformation to a (humanized) type 1 canonical conformation by studying the effect of selected mutations to the amino acid sequence of the H1, H2, and proximal residues. We use all-atomistic, explicit-solvent, biased molecular dynamic simulations to simulate the wild-type and mutant loops in a prefolded framework. We thus find mutants with increasing propensity to form a stable type 1 canonical conformation of the H1 loop. Free energy landscapes reveal the existence of conformational isomers of the canonical conformation that may play a role in binding different antigenic surfaces. We also elucidate the approximate mechanism and kinetics of transitions between such conformational isomers by using a Markovian model. We find that a particular three-point mutant has the strongest thermodynamic propensity to form the H1 type 1 canonical structure but also to exhibit transitions between conformational isomers, while a different, more rigid three-point mutant has the strongest propensity to be kinetically trapped in such a canonical structure.

Role of Arginine 293 and Glutamine 288 in Communication between Catalytic and Allosteric Sites in Yeast Ribonucleotide Reductase

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

Ahmad, Md. Faiz; Kaushal, Prem Singh; Wan, Qun

2012-11-01

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 ({alpha}) that contains the catalytic site and RR2 ({beta}) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-typemore » enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-({beta},{gamma}-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.« less

Drug development and manufacturing

DOEpatents

Warner, Benjamin P.; McCleskey, T. Mark; Burrell, Anthony K.

2015-10-13

X-ray fluorescence (XRF) spectrometry has been used for detecting binding events and measuring binding selectivities between chemicals and receptors. XRF may also be used for estimating the therapeutic index of a chemical, for estimating the binding selectivity of a chemical versus chemical analogs, for measuring post-translational modifications of proteins, and for drug manufacturing.

A look at ligand binding thermodynamics in drug discovery.

PubMed

Claveria-Gimeno, Rafael; Vega, Sonia; Abian, Olga; Velazquez-Campoy, Adrian

2017-04-01

Drug discovery is a challenging endeavor requiring the interplay of many different research areas. Gathering information on ligand binding thermodynamics may help considerably in reducing the risk within a high uncertainty scenario, allowing early rejection of flawed compounds and pushing forward optimal candidates. In particular, the free energy, the enthalpy, and the entropy of binding provide fundamental information on the intermolecular forces driving such interaction. Areas covered: The authors review the current status and recent developments in the application of ligand binding thermodynamics in drug discovery. The thermodynamic binding profile (Gibbs energy, enthalpy, and entropy of binding) can be used for lead selection and optimization (binding enthalpy, selectivity, and adaptability). Expert opinion: Binding thermodynamics provides fundamental information on the forces driving the formation of the drug-target complex. It has been widely accepted that binding thermodynamics may be used as a decision criterion along the ligand optimization process in drug discovery and development. In particular, the binding enthalpy may be used as a guide when selecting and optimizing compounds over a set of potential candidates. However, this has been recently called into question by arguing certain difficulties and in the light of certain experimental examples.

Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: binding of phosphonamidate to the S₁'-pocket of thermolysin.

PubMed

Englert, L; Biela, A; Zayed, M; Heine, A; Hangauer, D; Klebe, G

2010-11-01

Prerequisite for the design of tight binding protein inhibitors and prediction of their properties is an in-depth understanding of the structural and thermodynamic details of the binding process. A series of closely related phosphonamidates was studied to elucidate the forces underlying their binding affinity to thermolysin. The investigated inhibitors are identical except for the parts penetrating into the hydrophobic S₁'-pocket. A correlation of structural, kinetic and thermodynamic data was carried out by X-ray crystallography, kinetic inhibition assay and isothermal titration calorimetry. Binding affinity increases with larger ligand hydrophobic P₁'-moieties accommodating the S₁'-pocket. Surprisingly, larger P₁'-side chain modifications are accompanied by an increase in the enthalpic contribution to binding. In agreement with other studies, it is suggested that the release of largely disordered waters from an imperfectly hydrated pocket results in an enthalpically favourable integration of these water molecules into bulk water upon inhibitor binding. This enthalpically favourable process contributes more strongly to the binding energetics than the entropy increase resulting from the release of water molecules from the S₁'-pocket or the formation of apolar interactions between protein and inhibitor. Displacement of highly disordered water molecules from a rather imperfectly hydrated and hydrophobic specificity pocket can reveal an enthalpic signature of inhibitor binding. Copyright © 2010 Elsevier B.V. All rights reserved.

Cooperative Binding of Cyclodextrin Dimers to Isoflavone Analogues Elucidated by Free Energy Calculations.

PubMed

Zhang, Haiyang; Tan, Tianwei; Hetényi, Csaba; Lv, Yongqin; van der Spoel, David

2014-04-03

Dimerization of cyclodextrin (CD) molecules is an elementary step in the construction of CD-based nanostructured materials. Cooperative binding of CD cavities to guest molecules facilitates the dimerization process and, consequently, the overall stability and assembly of CD nanostructures. In the present study, all three dimerization modes (head-to-head, head-to-tail, and tail-to-tail) of β-CD molecules and their binding to three isoflavone drug analogues (puerarin, daidzin, and daidzein) were investigated in explicit water surrounding using molecular dynamics simulations. Total and individual contributions from the binding partners and solvent environment to the thermodynamics of these binding reactions are quantified in detail using free energy calculations. Cooperative drug binding to two CD cavities gives an enhanced binding strength for daidzin and daidzein, whereas for puerarin no obvious enhancement is observed. Head-to-head dimerization yields the most stable complexes for inclusion of the tested isoflavones (templates) and may be a promising building block for construction of template-stabilized CD nanostructures. Compared to the case of CD monomers, the desolvation of CD dimers and entropy changes upon complexation prove to be influential factors of cooperative binding. Our results shed light on key points of the design of CD-based supramolecular assemblies. We also show that structure-based calculation of binding thermodynamics can quantify stabilization caused by cooperative effects in building blocks of nanostructured materials.

Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

PubMed

Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

2016-07-05

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

Elucidating Molecular Motion through Structural and Dynamic Filters of Energy-Minimized Conformer Ensembles

PubMed Central

2015-01-01

Complex RNA structures are constructed from helical segments connected by flexible loops that move spontaneously and in response to binding of small molecule ligands and proteins. Understanding the conformational variability of RNA requires the characterization of the coupled time evolution of interconnected flexible domains. To elucidate the collective molecular motions and explore the conformational landscape of the HIV-1 TAR RNA, we describe a new methodology that utilizes energy-minimized structures generated by the program “Fragment Assembly of RNA with Full-Atom Refinement (FARFAR)”. We apply structural filters in the form of experimental residual dipolar couplings (RDCs) to select a subset of discrete energy-minimized conformers and carry out principal component analyses (PCA) to corroborate the choice of the filtered subset. We use this subset of structures to calculate solution T1 and T1ρ relaxation times for 13C spins in multiple residues in different domains of the molecule using two simulation protocols that we previously published. We match the experimental T1 times to within 2% and the T1ρ times to within less than 10% for helical residues. These results introduce a protocol to construct viable dynamic trajectories for RNA molecules that accord well with experimental NMR data and support the notion that the motions of the helical portions of this small RNA can be described by a relatively small number of discrete conformations exchanging over time scales longer than 1 μs. PMID:24479561

Structure of the mouse sex peptide pheromone ESP1 reveals a molecular basis for specific binding to the class C G-protein-coupled vomeronasal receptor.

PubMed

Yoshinaga, Sosuke; Sato, Toru; Hirakane, Makoto; Esaki, Kaori; Hamaguchi, Takashi; Haga-Yamanaka, Sachiko; Tsunoda, Mai; Kimoto, Hiroko; Shimada, Ichio; Touhara, Kazushige; Terasawa, Hiroaki

2013-05-31

Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.

Insights into the surface topology of polyhydroxyalkanoate synthase: self-assembly of functionalized inclusions.

PubMed

Hooks, David O; Rehm, Bernd H A

2015-10-01

The polyhydroxyalkanoate (PHA) synthase catalyzes the synthesis of PHA and remains attached to the hydrophobic PHA inclusions it creates. Although this feature is actively exploited to generate functionalized biobeads via protein engineering, little is known about the structure of the PHA synthase. Here, the surface topology of Ralstonia eutropha PHA synthase was probed to inform rational protein engineering toward the production of functionalized PHA beads. Surface-exposed residues were detected by conjugating biotin to inclusion-bound PHA synthase and identifying the biotin-conjugated lysine and cysteine residues using peptide fingerprinting analysis. The identified sites (K77, K90, K139, C382, C459, and K518) were investigated as insertion sites for the generation of new protein fusions. Insertions of FLAG epitopes into exposed sites K77, K90, K139, and K518 were tolerated, retaining >65 % of in vivo activity. Sites K90, K139, and K518 were also tested by insertion of the immunoglobulin G (IgG)-binding domain (ZZ), successfully producing PHA inclusions able to bind human IgG in vitro. Although simultaneous insertions of the ZZ domain into two sites was permissive, insertion at all three lysine sites inactivated the synthase. The K90/K139 double ZZ insertion had the optimum IgG-binding capacity of 16 mg IgG/g wet PHA beads and could selectively purify the IgG fraction from human serum. Overall, this study identified surface-exposed flexible regions of the PHA synthase which either tolerate protein/peptide insertions or are critical for protein function. This further elucidates the structure and function of PHA synthase and provides new opportunities for generating functionalized PHA biobeads.

The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G-A cross-linking PBD-duocarmycin dimers.

PubMed

Jackson, Paul J M; Rahman, Khondaker M; Thurston, David E

2017-01-01

The pyrrolobenzodiazepine (PBD) and duocarmycin families are DNA-interactive agents that covalently bond to guanine (G) and adenine (A) bases, respectively, and that have been joined together to create synthetic dimers capable of cross-linking G-G, A-A, and G-A bases. Three G-A alkylating dimers have been reported in publications to date, with defined DNA-binding sites proposed for two of them. In this study we have used molecular dynamics simulations to elucidate preferred DNA-binding sites for the three published molecular types. For the PBD-CPI dimer UTA-6026 (1), our simulations correctly predicted its favoured binding site (i.e., 5'-C(G)AATTA-3') as identified by DNA cleavage studies. However, for the PBD-CI molecule ('Compound 11', 3), we were unable to reconcile the results of our simulations with the reported preferred cross-linking sequence (5'-ATTTTCC(G)-3'). We found that the molecule is too short to span the five base pairs between the A and G bases as claimed, but should target instead a sequence such as 5'-ATTTC(G)-3' with two less base pairs between the reacting G and A residues. Our simulation results for this hybrid dimer are also in accord with the very low interstrand cross-linking and in vitro cytotoxicity activities reported for it. Although a preferred cross-linking sequence was not reported for the third hybrid dimer ('27eS', 2), our simulations predict that it should span two base pairs between covalently reacting G and A bases (e.g., 5'-GTAT(A)-3'). Copyright © 2016. Published by Elsevier Ltd.

Uranium(VI) Binding Forms in Selected Human Body Fluids: Thermodynamic Calculations versus Spectroscopic Measurements.

PubMed

Osman, Alfatih A A; Geipel, Gerhard; Barkleit, Astrid; Bernhard, Gert

2015-02-16

Human exposure to uranium increasingly becomes a subject of interest in many scientific disciplines such as environmental medicine, toxicology, and radiation protection. Knowledge about uranium chemical binding forms(speciation) in human body fluids can be of great importance to understand not only its biokinetics but also its relevance in risk assessment and in designing decorporation therapy in the case of accidental overexposure. In this study, thermodynamic calculations of uranium speciation in relevant simulated and original body fluids were compared with spectroscopic data after ex-situ uranium addition. For the first time, experimental data on U(VI) speciation in body fluids (saliva, sweat, urine) was obtained by means of cryogenic time-resolved laser-induced fluorescence spectroscopy (cryo-TRLFS) at 153 K. By using the time dependency of fluorescence decay and the band positions of the emission spectra, various uranyl complexes were demonstrated in the studied samples. The variations of the body fluids in terms of chemical composition, pH, and ionic strength resulted in different binding forms of U(VI). The speciation of U(VI) in saliva and in urine was affected by the presence of bioorganic ligands, whereas in sweat, the distribution depends mainly on inorganic ligands. We also elucidated the role of biological buffers, i.e., phosphate (H(2)PO(4−)/HPO(4)(2−)) on U(VI) distribution, and the system Ca(2+)/UO(2)(2+)/PO(4)(3−) was discussed in detail in both saliva and urine. The theoretical speciation calculations of the main U(VI) species in the investigated body fluids were significantly consistent with the spectroscopic data. Laser fluorescence spectroscopy showed success and reliability for direct determination of U(VI) in such biological matrices with the possibility for further improvement.

Histone deacetylase and GATA-binding factor 6 regulate arterial remodeling in angiotensin II-induced hypertension.

PubMed

Kim, Gwi Ran; Cho, Soo-Na; Kim, Hyung-Seok; Yu, Seon Young; Choi, Sin Young; Ryu, Yuhee; Lin, Ming Quan; Jin, Li; Kee, Hae Jin; Jeong, Myung Ho

2016-11-01

Histone deacetylase (HDAC) inhibitors have been reported to improve essential and secondary hypertension. However, the specific HDAC that might serve as a therapeutic target and the associated upstream and downstream molecules involved in regulating hypertension remain unknown. Our study was aimed at investigating whether a selective inhibitor of class II HDAC (MC1568) modulates hypertension, elucidating the underlying mechanism. Hypertension was established by administering angiotensin II (Ang II) to mice before treatment with MC1568. SBP was measured. Treatment with MC1568 reduced elevated SBP; attenuated arterial remodeling in the kidney's small arteries and thoracic aorta; and inhibited cell cycle regulatory gene expression, vascular smooth muscle cell (VSMC) proliferation, DNA synthesis, and VSMC hypertrophy in vivo and in vitro. Ang II enhanced the expression of phosphorylated HDAC4 and GATA-binding factor 6 (GATA6) proteins, which were specifically localized in the cytoplasm of cells in the arteries of kidneys and in aortas. Forced expression and knockdown of HDAC4 increased and decreased, respectively, the proliferation and expression of cell cycle genes in VSMCs. GATA6, a newly described binding partner of HDAC4, markedly enhanced the size and number of VSMCs. Calcium/calmodulin-dependent kinase IIα (CaMKIIα), but not HDAC4, translocated from the nucleus to the cytoplasm in response to Ang II. CaMKIIα and protein kinase D1 were associated with VSMC hypertrophy and hyperplasia via direct interaction with HDAC4. MC1568 treatment weakened the association between HDAC4 and CaMKIIα. These results suggest that class II HDAC inhibition attenuates hypertension by negatively regulating VSMC hypertrophy and hyperplasia via the CaMKIIα/protein kinase D1/HDAC4/GATA6 pathway.

Differential effects of clinically used derivatives and metabolites of artemisinin in the activation of constitutive androstane receptor isoforms

PubMed Central

Burk, O; Piedade, R; Ghebreghiorghis, L; Fait, JT; Nussler, AK; Gil, JP; Windshügel, B; Schwab, M

2012-01-01

BACKGROUND AND PURPOSE Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drug–drug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drug–drug interaction possibilities, the inducing properties of artemisinin metabolites should be considered. PMID:22577882

Hinge residue I174 is critical for proper dNTP selection by DNA polymerase beta.

PubMed

Yamtich, Jen; Starcevic, Daniela; Lauper, Julia; Smith, Elenoe; Shi, Idina; Rangarajan, Sneha; Jaeger, Joachim; Sweasy, Joann B

2010-03-23

DNA polymerase beta (pol beta) is the key gap-filling polymerase in base excision repair, the DNA repair pathway responsible for repairing up to 20000 endogenous lesions per cell per day. Pol beta is also widely used as a model polymerase for structure and function studies, and several structural regions have been identified as being critical for the fidelity of the enzyme. One of these regions is the hydrophobic hinge, a network of hydrophobic residues located between the palm and fingers subdomains. Previous work by our lab has shown that hinge residues Y265, I260, and F272 are critical for polymerase fidelity by functioning in discrimination of the correct from incorrect dNTP during ground state binding. Our work aimed to elucidate the role of hinge residue I174 in polymerase fidelity. To study this residue, we conducted a genetic screen to identify mutants with a substitution at residue I174 that resulted in a mutator polymerase. We then chose the mutator mutant I174S for further study and found that it follows the same general kinetic pathway as and has an overall protein folding similar to that of wild-type (WT) pol beta. Using single-turnover kinetic analysis, we found that I174S exhibits decreased fidelity when inserting a nucleotide opposite a template base G, and this loss of fidelity is due primarily to a loss of discrimination during ground state dNTP binding. Molecular dynamics simulations show that mutation of residue I174 to serine results in an overall tightening of the hinge region, resulting in aberrant protein dynamics and fidelity. These results point to the hinge region as being critical in the maintenance of the proper geometry of the dNTP binding pocket.

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies.

PubMed

Majumdar, Ritankar; Railkar, Reema; Dighe, Rajan R

2011-11-01

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions. Copyright © 2011 Wiley-Liss, Inc.

N- vs. C-Domain Selectivity of Catalytic Inactivation of Human Angiotensin Converting Enzyme by Lisinopril-Coupled Transition Metal Chelates

PubMed Central

Hocharoen, Lalintip; Joyner, Jeff C.; Cowan, J. A.

2014-01-01

The N- and C-terminal domains of human somatic Angiotensin I Converting Enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates were tested for both reversible binding and irreversible catalytic inactivation of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of the M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and orientation factors (double-filter effect). PMID:24228790

N- versus C-domain selectivity of catalytic inactivation of human angiotensin converting enzyme by lisinopril-coupled transition metal chelates.

PubMed

Hocharoen, Lalintip; Joyner, Jeff C; Cowan, J A

2013-12-27

The N- and C-terminal domains of human somatic angiotensin I converting enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates was tested for both reversible binding and irreversible catalytic inactivation of each domain of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and catalytic factors (double-filter effect).

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

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

Derebe, Mehabaw G.; Sauer, David B.; Zeng, Weizhong

2015-11-30

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K{sup +} channel selectivity filters. Combined with single channel electrophysiology, we show that only themore » channel with four ion binding sites is K{sup +} selective, whereas those with two or three are nonselective and permeate Na{sup +} and K{sup +} equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K{sup +} channels is essential for highly selective and efficient permeation of K{sup +} ions.« less

The structure and binding mode of citrate in the stabilization of gold nanoparticles

NASA Astrophysics Data System (ADS)

Al-Johani, Hind; Abou-Hamad, Edy; Jedidi, Abdesslem; Widdifield, Cory M.; Viger-Gravel, Jasmine; Sangaru, Shiv Shankar; Gajan, David; Anjum, Dalaver H.; Ould-Chikh, Samy; Hedhili, Mohamed Nejib; Gurinov, Andrei; Kelly, Michael J.; El Eter, Mohamad; Cavallo, Luigi; Emsley, Lyndon; Basset, Jean-Marie

2017-09-01

Elucidating the binding mode of carboxylate-containing ligands to gold nanoparticles (AuNPs) is crucial to understand their stabilizing role. A detailed picture of the three-dimensional structure and coordination modes of citrate, acetate, succinate and glutarate to AuNPs is obtained by 13C and 23Na solid-state NMR in combination with computational modelling and electron microscopy. The binding between the carboxylates and the AuNP surface is found to occur in three different modes. These three modes are simultaneously present at low citrate to gold ratios, while a monocarboxylate monodentate (1κO1) mode is favoured at high citrate:gold ratios. The surface AuNP atoms are found to be predominantly in the zero oxidation state after citrate coordination, although trace amounts of Auδ+ are observed. 23Na NMR experiments show that Na+ ions are present near the gold surface, indicating that carboxylate binding occurs as a 2e- L-type interaction for each oxygen atom involved. This approach has broad potential to probe the binding of a variety of ligands to metal nanoparticles.

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.

Molecular Mechanism Underlying the Entomotoxic Effect of Colocasia esculenta Tuber Agglutinin against Dysdercus cingulatus

PubMed Central

Roy, Amit; Das, Sampa

2015-01-01

Colocasia esculenta tuber agglutinin (CEA), a mannose binding lectin, exhibits insecticidal efficacy against different hemipteran pests. Dysdercus cingulatus, red cotton bug (RCB), has also shown significant susceptibility to CEA intoxication. However, the molecular basis behind such entomotoxicity of CEA has not been addressed adequately. The present study elucidates the mechanism of insecticidal efficacy of CEA against RCB. Confocal and scanning electron microscopic analyses documented CEA binding to insect midgut tissue, resulting in an alteration of perimicrovillar membrane (PMM) morphology. Internalization of CEA into insect haemolymph and ovary was documented by western blotting analyses. Ligand blot followed by mass spectrometric identification revealed the cognate binding partners of CEA as actin, ATPase and cytochrome P450. Deglycosylation and mannose inhibition assays indicated the interaction to probably be mannose mediated. Bioinformatic identification of putative glycosylation or mannosylation sites in the binding partners further supports the sugar mediated interaction. Correlating entomotoxicity of CEA with immune histological and binding assays to the insect gut contributes to a better understanding of the insecticidal potential of CEA and endorses its future biotechnological application.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

PubMed

Bai, Fang; Morcos, Faruck; Cheng, Ryan R; Jiang, Hualiang; Onuchic, José N

2016-12-13

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Design and Nuclear Magnetic Resonance (NMR) Structure Determination of the Second Extracellular Immunoglobulin Tyrosine Kinase A (TrkAIg2) Domain Construct for Binding Site Elucidation in Drug Discovery

PubMed Central

2014-01-01

The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein–ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a β-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF (1WWW.pdb), and the 1H–15N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution. PMID:25454499

Binding interaction of atorvastatin with bovine serum albumin: Spectroscopic methods and molecular docking

NASA Astrophysics Data System (ADS)

Wang, Qi; Huang, Chuan-ren; Jiang, Min; Zhu, Ying-yao; Wang, Jing; Chen, Jun; Shi, Jie-hua

2016-03-01

The interaction of atorvastatin with bovine serum albumin (BSA) was investigated using multi-spectroscopic methods and molecular docking technique for providing important insight into further elucidating the store and transport process of atorvastatin in the body and the mechanism of action and pharmacokinetics. The experimental results revealed that the fluorescence quenching mechanism of BSA induced atorvastatin was a combined dynamic and static quenching. The binding constant and number of binding site of atorvastatin with BSA under simulated physiological conditions (pH = 7.4) were 1.41 × 105 M- 1 and about 1 at 310 K, respectively. The values of the enthalpic change (ΔH0), entropic change (ΔS0) and Gibbs free energy (ΔG0) in the binding process of atorvastatin with BSA at 310 K were negative, suggesting that the binding process of atorvastatin and BSA was spontaneous and the main interaction forces were van der Waals force and hydrogen bonding interaction. Moreover, atorvastatin was bound into the subdomain IIA (site I) of BSA, resulting in a slight change of the conformation of BSA.

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

PubMed Central

Evers, R; Grummt, I

1995-01-01

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597036

Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

PubMed Central

Moroni, Elisabetta; Zhao, Huiping; Blagg, Brian S.J.; Colombo, Giorgio

2014-01-01

The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site. PMID:24397468

Interaction of antithrombin with sulfated, low molecular weight lignins: opportunities for potent, selective modulation of antithrombin function.

PubMed

Henry, Brian L; Connell, Justin; Liang, Aiye; Krishnasamy, Chandravel; Desai, Umesh R

2009-07-31

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891-31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80-87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.

Complex coacervation in charge complementary biopolymers: Electrostatic versus surface patch binding.

PubMed

Pathak, Jyotsana; Priyadarshini, Eepsita; Rawat, Kamla; Bohidar, H B

2017-12-01

In this review, a number of systems are described to demonstrate the effect of polyelectrolyte chain stiffness (persistence length) on the coacervation phenomena, after we briefly review the field. We consider two specific types of complexation/coacervation: in the first type, DNA is used as a fixed substrate binding to flexible polyions such as gelatin A, bovine serum albumin and chitosan (large persistence length polyelectrolyte binding to low persistence length biopolymer), and in the second case, different substrates such as gelatin A, bovine serum albumin, and chitosan were made to bind to a polyion gelatin B (low persistence length substrate binding to comparable persistence length polyion). Polyelectrolyte chain flexibility was found to have remarkable effect on the polyelectrolyte-protein complex coacervation. The competitive interplay of electrostatic versus surface patch binding (SPB) leading to associative interaction followed by complex coacervation between these biopolymers is elucidated. We modelled the SPB interaction in terms of linear combination of attractive and repulsive Coulombic forces with respect to the solution ionic strength. The aforesaid interactions were established via a universal phase diagram, considering the persistence length of polyion as the sole independent variable. Copyright © 2017 Elsevier B.V. All rights reserved.

Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea.

PubMed

Shuttleworth, Gillian; Fogg, Mark J; Kurpiewski, Michael R; Jen-Jacobson, Linda; Connolly, Bernard A

2004-03-26

Archaeal family B DNA polymerases contain a specialised pocket that binds tightly to template-strand uracil, causing the stalling of DNA replication. The mechanism of this unique "template-strand proof-reading" has been studied using equilibrium binding measurements, DNA footprinting, van't Hoff analysis and calorimetry. Binding assays have shown that the polymerase preferentially binds to uracil in single as opposed to double-stranded DNA. Tightest binding is observed using primer-templates that contain uracil four bases in front of the primer-template junction, corresponding to the observed stalling position. Ethylation interference analysis of primer-templates shows that the two phosphates, immediately flanking the uracil (NpUpN), are important for binding; contacts are also made to phosphates in the primer-strand. Microcalorimetry and van't Hoff analysis have given a fuller understanding of the thermodynamic parameters involved in uracil recognition. All the results are consistent with a "read-ahead" mechanism, in which the replicating polymerase scans the template, ahead of the replication fork, for the presence of uracil and halts polymerisation on detecting this base. Post-stalling events, serving to eliminate uracil, await full elucidation.

Cyclotides Insert into Lipid Bilayers to Form Membrane Pores and Destabilize the Membrane through Hydrophobic and Phosphoethanolamine-specific Interactions*

PubMed Central

Wang, Conan K.; Wacklin, Hanna P.; Craik, David J.

2012-01-01

Cyclotides are a family of plant-derived circular proteins with potential therapeutic applications arising from their remarkable stability, broad sequence diversity, and range of bioactivities. Their membrane-binding activity is believed to be a critical component of their mechanism of action. Using isothermal titration calorimetry, we studied the binding of the prototypical cyclotides kalata B1 and kalata B2 (and various mutants) to dodecylphosphocholine micelles and phosphoethanolamine-containing lipid bilayers. Although binding is predominantly an entropy-driven process, suggesting that hydrophobic forces contribute significantly to cyclotide-lipid complex formation, specific binding to the phosphoethanolamine-lipid headgroup is also required, which is evident from the enthalpic changes in the free energy of binding. In addition, using a combination of dissipative quartz crystal microbalance measurements and neutron reflectometry, we elucidated the process by which cyclotides interact with bilayer membranes. Initially, a small number of cyclotides bind to the membrane surface and then insert first into the outer membrane leaflet followed by penetration through the membrane and pore formation. At higher concentrations of cyclotides, destabilization of membranes occurs. Our results provide significant mechanistic insight into how cyclotides exert their bioactivities. PMID:23129773

Multiple and cooperative binding of fluorescence light-up probe thioflavin T with human telomere DNA G-quadruplex.

PubMed

Gabelica, Valérie; Maeda, Ryuichi; Fujimoto, Takeshi; Yaku, Hidenobu; Murashima, Takashi; Sugimoto, Naoki; Miyoshi, Daisuke

2013-08-20

Thioflavin T (ThT), a typical probe for protein fibrils, also binds human telomeric G-quadruplexes with a fluorescent light-up signal change and high specificity against DNA duplexes. Cell penetration and low cytotoxicity of fibril probes having been widely established, modifying ThT and other fibril probes is an attractive means of generating new G-quadruplex ligands. Thus, elucidating the binding mechanism is important for the design of new drugs and fluorescent probes based on ThT. Here, we investigated the binding mechanism of ThT with several variants of the human telomeric sequence in the presence of monovalent cations. Fluorescence titrations and electrospray ionization mass spectrometry (ESI-MS) analyses demonstrated that each G-quadruplex unit cooperatively binds to several ThT molecules. ThT brightly fluoresces when a single ligand is bound to the G-quadruplex and is quenched as ligand binding stoichiometry increases. Both the light-up signal and the dissociation constants are exquisitely sensitive to the base sequence and to the G-quadruplex structure. These results are crucial for the sensible design and interpretation of G-quadruplex detection assays using fluorescent ligands in general and ThT in particular.

Interaction of AIM with insulin-like growth factor-binding protein-4.

PubMed

You, Qiang; Wu, Yan; Yao, Nannan; Shen, Guannan; Zhang, Ying; Xu, Liangguo; Li, Guiying; Ju, Cynthia

2015-09-01

Apoptosis inhibitor of macrophages (AIM/cluster of differentiation 5 antigen-like/soluble protein α) has been shown to inhibit cellular apoptosis; however, the underlying molecular mechanism has not been elucidated. Using yeast two‑hybrid screening, the present study uncovered that AIM binds to insulin‑like growth factor binding protein‑4 (IGFBP‑4). AIM interaction with IGFBP‑4, as well as IGFBP‑2 and ‑3, but not with IGFBP‑1, ‑5 and ‑6, was further confirmed by co‑immunoprecipitation (co‑IP) using 293 cells. The binding activity and affinity between AIM and IGFBP‑4 in vitro were analyzed by co‑IP and biolayer interferometry. Serum depletion‑induced cellular apoptosis was attenuated by insulin‑like growth factor‑I (IGF‑I), and this effect was abrogated by IGFBP‑4. Of note, in the presence of AIM, the inhibitory effect of IGFBP‑4 on the anti‑apoptosis function of IGF‑I was attenuated, possibly through binding of AIM with IGFBP‑4. In conclusion, to the best of our knowledge, the present study provides the first evidence that AIM binds to IGFBP‑2, ‑3 and ‑4. The data suggest that this interaction may contribute to the mechanism of AIM-mediated anti-apoptosis function.

Interaction of AIM with insulin-like growth factor-binding protein-4

PubMed Central

YOU, QIANG; WU, YAN; YAO, NANNAN; SHEN, GUANNAN; ZHANG, YING; XU, LIANGGUO; LI, GUIYING; JU, CYNTHIA

2015-01-01

Apoptosis inhibitor of macrophages (AIM/cluster of differentiation 5 antigen-like/soluble protein α) has been shown to inhibit cellular apoptosis; however, the underlying molecular mechanism has not been elucidated. Using yeast two-hybrid screening, the present study uncovered that AIM binds to insulin-like growth factor binding protein-4 (IGFBP-4). AIM interaction with IGFBP-4, as well as IGFBP-2 and -3, but not with IGFBP-1, -5 and -6, was further confirmed by co-immunoprecipitation (co-IP) using 293 cells. The binding activity and affinity between AIM and IGFBP-4 in vitro were analyzed by co-IP and biolayer interferometry. Serum depletion-induced cellular apoptosis was attenuated by insulin-like growth factor-I (IGF-I), and this effect was abrogated by IGFBP-4. Of note, in the presence of AIM, the inhibitory effect of IGFBP-4 on the anti-apoptosis function of IGF-I was attenuated, possibly through binding of AIM with IGFBP-4. In conclusion, to the best of our knowledge, the present study provides the first evidence that AIM binds to IGFBP-2, -3 and -4. The data suggest that this interaction may contribute to the mechanism of AIM-mediated anti-apoptosis function. PMID:26135353

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains♦

PubMed Central

Tanaka, Hiroaki; Akagi, Ken-ichi; Oneyama, Chitose; Tanaka, Masakazu; Sasaki, Yuichi; Kanou, Takashi; Lee, Young-Ho; Yokogawa, Daisuke; Dobenecker, Marc-Werner; Nakagawa, Atsushi; Okada, Masato; Ikegami, Takahisa

2013-01-01

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain. PMID:23548896

Membrane surface engineering for protein separations: experiments and simulations.

PubMed

Liu, Zizhao; Du, Hongbo; Wickramasinghe, S Ranil; Qian, Xianghong

2014-09-09

A bisphosphonate derived ligand was successfully synthesized and grafted from the surface of regenerated cellulose membrane using atom transfer radical polymerization (ATRP) for protein separations. This ligand has a remarkable affinity for arginine (Arg) residues on protein surface. Hydrophilic residues N-(2-hydroxypropyl) methacrylamide (HPMA) was copolymerized to enhance the flexibility of the copolymer ligand and further improve specific protein adsorption. The polymerization of bisphosphonate derivatives was successful for the first time using ATRP. Static and dynamic binding capacities were determined for binding and elution of Arg rich lysozyme. The interaction mechanism between the copolymer ligand and lysozyme was elucidated using classical molecular dynamics (MD) simulations.

Biomolecular characterization of glass surfaces

NASA Astrophysics Data System (ADS)

Clare, Alexis G.; Hall, Matthew M.; Korwin-Edson, Michelle L.; Goldstein, Alan H.

2003-08-01

This paper introduces the concept of biomolecular characterization of inorganic surfaces. The choice of biomolecule is discussed followed by techniques that can be used to analyse the quantity of bound species, strength of binding, the nature of binding sites, conformational changes and the layer morphology. The prospects of modelling this data using a combination of molecular dynamics simulation and protein structural modelling and the correlation to measured data are outlined. The studies described in this paper are directed toward assessing the feasibility of biomolecular characterization, however, the data collected in the process are designed to also help elucidate our understanding of the interaction between biomolecular species and inorganic materials interfaces.

Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

PubMed

Liau, Nicholas P D; Laktyushin, Artem; Babon, Jeffrey J

2017-01-01

Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

PubMed

Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie; Sha, Fern; Pojer, Florence; Koide, Akiko; Seeliger, Markus; Koide, Shohei; Hantschel, Oliver

2017-05-05

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

DOE PAGES

Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco; ...

2015-12-24

Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsinmore » kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.« less

Conformational selection in a protein-protein interaction revealed by dynamic pathway analysis

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

Chakrabarti, Kalyan S.; Agafonov, Roman V.; Pontiggia, Francesco

Molecular recognition plays a central role in biology, and protein dynamics has been acknowledged to be important in this process. However, it is highly debated whether conformational changes happen before ligand binding to produce a binding-competent state (conformational selection) or are caused in response to ligand binding (induced fit). Proposals for both mechanisms in protein/protein recognition have been primarily based on structural arguments. However, the distinction between them is a question of the probabilities of going via these two opposing pathways. Here we present a direct demonstration of exclusive conformational selection in protein/protein recognition by measuring the flux for rhodopsinmore » kinase binding to its regulator recoverin, an important molecular recognition in the vision system. Using NMR spectroscopy, stopped-flow kinetics and isothermal titration calorimetry we show that recoverin populates a minor conformation in solution that exposes a hydrophobic binding pocket responsible for binding rhodopsin kinase. Lastly, protein dynamics in free recoverin limits the overall rate of binding.« less

Insight into the enzyme-inhibitor interactions of the first experimentally determined human aromatase.

PubMed

Punetha, Ankita; Shanmugam, Karthi; Sundar, Durai

2011-04-01

Aromatase is an important pharmacological target in the anti-cancer therapy as the intratumoral aromatase is the source of local estrogen production in breast cancer tissues. Suppression of estrogen biosynthesis by aromatase inhibition represents an effective approach for the treatment of hormone-sensitive breast cancer. Because of the membrane-bound character and heme-binding instability, no crystal structure of aromatase was reported for a long time, until recently when crystal structure of human placental aromatase cytochrome P450 in complex with androstenedione was deposited in PDB. The present study is towards understanding the structural and functional characteristics of aromatase to address unsolved mysteries about this enzyme and elucidate the exact mode of binding of aromatase inhibitors. We have performed molecular docking simulation with twelve different inhibitors (ligands), which includes four FDA approved drugs; two flavonoids; three herbal compounds and three compounds having biphenyl motif with known IC(50) values into the active site of the human aromatase enzyme. All ligands showed favorable interactions and most of them seemed to interact to hydrophobic amino acids Ile133, Phe134, Phe221, Trp224, Ala306, Val370, Val373, Met374 and Leu477 and hydrophilic Arg115 and neutral Thr310 residues. The elucidation of the actual structure-function relationship of aromatase and the exact binding mode described in this study will be of significant interest as its inhibitors have shown great promise in fighting breast cancer.

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

PubMed

Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

2007-05-15

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

Identification of chondrocyte-binding peptides by phage display.

PubMed

Cheung, Crystal S F; Lui, Julian C; Baron, Jeffrey

2013-07-01

As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. Copyright © 2013 Orthopaedic Research Society.

Photoactivable antibody binding protein: site-selective and covalent coupling of antibody.

PubMed

Jung, Yongwon; Lee, Jeong Min; Kim, Jung-won; Yoon, Jeongwon; Cho, Hyunmin; Chung, Bong Hyun

2009-02-01

Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.

NMR structural studies of the supramolecular adducts between a liver cytosolic bile acid binding protein and gadolinium(III)-chelates bearing bile acids residues: molecular determinants of the binding of a hepatospecific magnetic resonance imaging contrast agent.

PubMed

Assfalg, Michael; Gianolio, Eliana; Zanzoni, Serena; Tomaselli, Simona; Russo, Vito Lo; Cabella, Claudia; Ragona, Laura; Aime, Silvio; Molinari, Henriette

2007-11-01

The binding affinities of a selected series of Gd(III) chelates bearing bile acid residues, potential hepatospecific MRI contrast agents, to a liver cytosolic bile acid transporter, have been determined through relaxivity measurements. The Ln(III) complexes of compound 1 were selected for further NMR structural analysis aimed at assessing the molecular determinants of binding. A number of NMR experiments have been carried out on the bile acid-like adduct, using both diamagnetic Y(III) and paramagnetic Gd(III) complexes, bound to a liver bile acid binding protein. The identified protein "hot spots" defined a single binding site located at the protein portal region. The presented findings will serve in a medicinal chemistry approach for the design of hepatocytes-selective gadolinium chelates for liver malignancies detection.

Engineering Tocopherol Selectivity in α-TTP: A Combined In Vitro/In Silico Study

PubMed Central

Helbling, Rachel E.; Aeschimann, Walter; Simona, Fabio; Stocker, Achim; Cascella, Michele

2012-01-01

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of -tocopherol transfer protein (-TTP) towards -tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for -tocopherol to -TTP 8.262.13 kcal mol lower than that of -tocopherol. Our calculations show that -tocopherol binds to -TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of -TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to -tocopherol, with striking structural similarities to the wild-type--tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of -TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a -tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for -tocopherol selection in omnivorous animals. PMID:23152872

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

Force fields for describing the solution-phase synthesis of shape-selective metal nanoparticles

NASA Astrophysics Data System (ADS)

Zhou, Ya; Al-Saidi, Wissam; Fichthorn, Kristen

2013-03-01

Polyvinylpyrrolidone (PVP) and polyethylene oxide (PEO) are structure-directing agents that exhibit different performance in the polyol synthesis of Ag nanostructures. The success of these structure-directing agents in selective nanostructure synthesis is often attributed to their selective binding to Ag(100) facets. We use first-principles, density-functional theory (DFT) calculations in a vacuum environment to show that PVP has a stronger preference to bind to Ag(100) than to Ag(111), whereas PEO exhibits much weaker selectivity. To understand the role of solvent in the surface-sensitive binding, we develop classical force fields to describe the interactions of the structure-directing (PVP and PEO) and solvent (ethylene glycol) molecules with various Ag substrates. We parameterize the force fields through force-and-energy matching to DFT results using simulated annealing. We validate the force fields by comparisons to DFT and experimental binding energies. Our force fields reproduce the surface-sensitive binding predicted by DFT calculations. Molecular dynamics simulations based on these force fields can be used to reveal the role of solvent, polymer chain length, and polymer concentration in the selective synthesis of Ag nanostructures.

Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III

PubMed Central

Bezerra, Gustavo A.; Dobrovetsky, Elena; Viertlmayr, Roland; Dong, Aiping; Binter, Alexandra; Abramić, Marija; Macheroux, Peter; Dhe-Paganon, Sirano; Gruber, Karl

2012-01-01

Opioid peptides are involved in various essential physiological processes, most notably nociception. Dipeptidyl peptidase III (DPP III) is one of the most important enkephalin-degrading enzymes associated with the mammalian pain modulatory system. Here we describe the X-ray structures of human DPP III and its complex with the opioid peptide tynorphin, which rationalize the enzyme's substrate specificity and reveal an exceptionally large domain motion upon ligand binding. Microcalorimetric analyses point at an entropy-dominated process, with the release of water molecules from the binding cleft (“entropy reservoir”) as the major thermodynamic driving force. Our results provide the basis for the design of specific inhibitors that enable the elucidation of the exact role of DPP III and the exploration of its potential as a target of pain intervention strategies. PMID:22493238

Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

PubMed

Timr, Štěpán; Kadlec, Jan; Srb, Pavel; Ollila, O H Samuli; Jungwirth, Pavel

2018-04-05

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

Computational study of small molecule binding for both tethered and free conditions

PubMed Central

2010-01-01

Using a calix[4]arene-benzene complex as a test system we compare the potential of mean force for when the calix[4]arene is tethered versus free. When the complex is in vacuum our results show that the difference between tethered and free is primarily due to the entropic contribution to the potential of mean force resulting in a significant binding free energy difference of 6.6 kJ/mol. By contrast, when the complex is in water our results suggest that there is no appreciable difference between tethered and free. This study elucidates the roles of entropy and enthalpy for this small molecule system and emphasizes the point that tethering the receptor has the potential to dramatically impact the binding properties. These findings should be taken into consideration when using calixarene molecules in nanosensor design. PMID:20369865

Surface Plasmon Resonance Study of the Binding of PEO-PPO-PEO Triblock Copolymer and PEO Homopolymer to Supported Lipid Bilayers.

PubMed

Kim, Mihee; Vala, Milan; Ertsgaard, Christopher T; Oh, Sang-Hyun; Lodge, Timothy P; Bates, Frank S; Hackel, Benjamin J

2018-06-12

Poloxamer 188 (P188), a poly(ethylene oxide)- b-poly(propylene oxide)- b-poly(ethylene oxide) triblock copolymer, protects cell membranes against various external stresses, whereas poly(ethylene oxide) (PEO; 8600 g/mol) homopolymer lacks protection efficacy. As part of a comprehensive effort to elucidate the protection mechanism, we used surface plasmon resonance (SPR) to obtain direct evidence of binding of the polymers onto supported lipid bilayers. Binding kinetics and coverage of P188 and PEO were examined and compared. Most notably, PEO exhibited membrane association comparable to that of P188, evidenced by comparable association rate constants and coverage. This result highlights the need for additional mechanistic understanding beyond simple membrane association to explain the differential efficacy of P188 in therapeutic applications.

Determination of copper binding in Pseudomonas putida CZ1 by chemical modifications and X-ray absorption spectroscopy.

PubMed

Chen, XinCai; Shi, JiYan; Chen, YingXu; Xu, XiangHua; Chen, LiTao; Wang, Hui; Hu, TianDou

2007-03-01

Previously performed studies have shown that Pseudomonas putida CZ1 biomass can bind an appreciable amount of Cu(II) and Zn(II) ions from aqueous solutions. The mechanisms of Cu- and Zn-binding by P. putida CZ1 were ascertained by chemical modifications of the biomass followed by Fourier transform infrared and X-ray absorption spectroscopic analyses of the living or nonliving cells. A dramatic decrease in Cu(II)- and Zn(II)-binding resulted after acidic methanol esterification of the nonliving cells, indicating that carboxyl functional groups play an important role in the binding of metal to the biomaterial. X-ray absorption spectroscopy was used to determine the speciation of Cu ions bound by living and nonliving cells, as well as to elucidate which functional groups were involved in binding of the Cu ions. The X-ray absorption near-edge structure spectra analysis showed that the majority of the Cu was bound in both samples as Cu(II). The fitting results of Cu K-edge extended X-ray absorption fine structure spectra showed that N/O ligands dominated in living and nonliving cells. Therefore, by combining different techniques, our results indicate that carboxyl functional groups are the major ligands responsible for the metal binding in P. putida CZ1.

Solution Binding and Structural Analyses Reveal Potential Multidrug Resistance Functions for SAV2435 and CTR107 and Other GyrI-like Proteins.

PubMed

Moreno, Andrew; Froehlig, John R; Bachas, Sharrol; Gunio, Drew; Alexander, Teressa; Vanya, Aaron; Wade, Herschel

2016-08-30

Multidrug resistance (MDR) refers to the acquired ability of cells to tolerate a broad range of toxic compounds. One mechanism cells employ is to increase the level of expression of efflux pumps for the expulsion of xenobiotics. A key feature uniting efflux-related mechanisms is multidrug (MD) recognition, either by efflux pumps themselves or by their transcriptional regulators. However, models describing MD binding by MDR effectors are incomplete, underscoring the importance of studies focused on the recognition elements and key motifs that dictate polyspecific binding. One such motif is the GyrI-like domain, which is found in several MDR proteins and is postulated to have been adapted for small-molecule binding and signaling. Here we report the solution binding properties and crystal structures of two proteins containing GyrI-like domains, SAV2435 and CTR107, bound to various ligands. Furthermore, we provide a comparison with deposited crystal structures of GyrI-like proteins, revealing key features of GyrI-like domains that not only support polyspecific binding but also are conserved among GyrI-like domains. Together, our studies suggest that GyrI-like domains perform evolutionarily conserved functions connected to multidrug binding and highlight the utility of these types of studies for elucidating mechanisms of MDR.

Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing

PubMed Central

Kisseleva, Natalia; Kraut, Stefanie; Jäschke, Andres; Schiemann, Olav

2007-01-01

In ribozyme catalysis, metal ions are generally known to make structural and∕or mechanistic contributions. The catalytic activity of a previously described Diels-Alderase ribozyme was found to depend on the concentration of divalent metal ions, and crystallographic data revealed multiple binding sites. Here, we elucidate the interactions of this ribozyme with divalent metal ions in solution using electron paramagnetic resonance (EPR) spectroscopy. Manganese ion titrations revealed five high-affinity Mn2+ binding sites with an upper Kd of 0.6±0.2 μM. In order to characterize each binding site individually, EPR-silent Cd2+ ions were used to saturate the other binding sites. This cadmium-induced EPR silencing showed that the Mn2+ binding sites possess different affinities. In addition, these binding sites could be assigned to three different types, including innersphere, outersphere, and a Mn2+ dimer. Based on simulations, the Mn2+-Mn2+ distance within the dimer was found to be ∼6 Å, which is in good agreement with crystallographic data. The EPR-spectroscopic characterization reveals no structural changes upon addition of a Diels-Alder product, supporting the concept of a preorganized catalytic pocket in the Diels-Alder ribozyme and the structural role of these ions. PMID:19404418

Probing the Characterization of the Interaction of Aflatoxins B1 and G1 with Calf Thymus DNA In Vitro

PubMed Central

Ma, Liang; Wang, Jiaman; Zhang, Yuhao

2017-01-01

The binding characterization of aflatoxins with calf thymus DNA (ctDNA) under physiological conditions was investigated. Multispectroscopic techniques, ctDNA melting, viscosity measurements, and molecular docking techniques were employed to elucidate the binding mechanism of the aflatoxins with DNA. The fluorescence results indicated that both aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) bound to the ctDNA, forming complexes through hydrogen bonding. The binding constants of AFB1 and AFG1 with ctDNA reached up to 103 L·mol−1 and 104 L·mol−1, respectively, and AFG1 exhibited a higher binding propensity than that of AFB1. Furthermore, both AFB1 and AFG1 bound to the ctDNA through groove binding, as evidenced by the results of the spectroscopic, iodide quenching effect, viscosity, and ctDNA melting measurements. Changes in the circular dichroism signal manifested that both AFB1 and AFG1 induced an increase in the right-handed helicity, but only minimally influenced the base stacking of the DNA. A molecular docking study of the aflatoxin’s binding with the DNA revealed a groove binding mode, which was driven mainly by hydrogen bonding. This study of aflatoxin–ctDNA interaction may provide novel insights into the toxicological effect of the mycotoxins. PMID:28671585

X-ray Crystal Structures Elucidate the Nucleotidyl Transfer Reaction of Transcript Initiation Using Two Nucleotides

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

M Gleghorn; E Davydova; R Basu

2011-12-31

We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groupsmore » of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.« less

Receptor-ligand binding sites and virtual screening.

PubMed

Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

2006-01-01

Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

Initiation of Phage Infection by Partial Unfolding and Prolyl Isomerization*♦

PubMed Central

Hoffmann-Thoms, Stephanie; Weininger, Ulrich; Eckert, Barbara; Jakob, Roman P.; Koch, Johanna R.; Balbach, Jochen; Schmid, Franz X.

2013-01-01

Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time. PMID:23486474

Molecular insights into the binding of phosphoinositides to the TH domain region of TIPE proteins.

PubMed

Antony, Priya; Baby, Bincy; Vijayan, Ranjit

2016-11-01

Phosphatidylinositols and their phosphorylated derivatives, phosphoinositides, play a central role in regulating diverse cellular functions. These phospholipids have been shown to interact with the hydrophobic TH domain of the tumor necrosis factor (TNF)-α-induced protein 8 (TIPE) family of proteins. However, the precise mechanism of interaction of these lipids is unclear. Here we report the binding mode and interactions of these phospholipids in the TH domain, as elucidated using molecular docking and simulations. Results indicate that phosphoinositides bind to the TH domain in a similar way by inserting their lipid tails in the hydrophobic cavity. The exposed head group is stabilized by interactions with critical positively charged residues on the surface of these proteins. Further MD simulations confirmed the dynamic stability of these lipids in the TH domain. This computational analysis thus provides insight into the binding mode of phospholipids in the TH domain of the TIPE family of proteins. Graphical abstract A phosphoinositide (phosphatidylinositol 4-phosphate; PtdIns4P) docked to TIPE2.

Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

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

Schlunzen, Frank; Zarivach, Raz; Harms, Jörg

2009-10-07

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavitymore » and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.« less

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

PubMed

Ganesan, Lakshmi; Buchwald, Peter

2013-04-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

PubMed Central

Ganesan, Lakshmi; Buchwald, Peter

2013-01-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

Dynamics of human protein kinase Aurora A linked to drug selectivity

DOE PAGES

Pitsawong, Warintra; Buosi, Vanessa; Otten, Renee; ...

2018-06-14

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Auroramore » A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.« less

Dynamics of human protein kinase Aurora A linked to drug selectivity

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

Pitsawong, Warintra; Buosi, Vanessa; Otten, Renee

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Auroramore » A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.« less

Phage diabody repertoires for selection of large numbers of bispecific antibody fragments.

PubMed

McGuinness, B T; Walter, G; FitzGerald, K; Schuler, P; Mahoney, W; Duncan, A R; Hoogenboom, H R

1996-09-01

Methods for the generation of large numbers of different bispecific antibodies are presented. Cloning strategies are detailed to create repertoires of bispecific diabody molecules with variability on one or both of the antigen binding sites. This diabody format, when combined with the power of phage display technology, allows the generation and analysis of thousands of different bispecific molecules. Selection for binding presumably also selects for more stable diabodies. Phage diabody libraries enable screening or selection of the best combination bispecific molecule with regards to affinity of binding, epitope recognition and pairing before manufacture of the best candidate.

Characterization of [3H]LS-3-134, a Novel Arylamide Phenylpiperazine D3 Dopamine Receptor Selective Radioligand

PubMed Central

Rangel-Barajas, Claudia; Malik, Maninder; Taylor, Michelle; Neve, Kim A.; Mach, Robert H.; Luedtke, Robert R.

2014-01-01

LS-3-134 is a substituted N-phenylpiperazine derivative that has been reported to exhibit a) high-affinity binding (Ki value 0.2 nM) at human D3 dopamine receptors, b) >100-fold D3 vs. D2 dopamine receptor subtype binding selectivity and c) low-affinity binding (Ki values >5,000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin-dependent activation of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [3H]-labeled LS-3-134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10-15 min at 37°C) and binds with high affinity to both human (Kd = 0.06 ± 0.01 nM) and rat (Kd = 0.2 ± 0.02 nM) D3 receptors expressed in HEK-293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [3H]LS-3-134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies we propose that [3H]LS-3-134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype. PMID:25041389

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

Bidlack, J.M.; Frey, D.K.; Seyed-Mozaffari, A.

The binding properties of 14{beta}-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM ({sup 3}H)(D-Ala{sup 2},(Me)Phe{sup 4},Gly(ol){supmore » 5})enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the {mu} binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The {mu} receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the {delta}-selective peptide ({sup 3}H)(D-penicillamine{sup 2},D-penicillamine{sup 5})enkephalin (DPDPE) and (-)-({sup 3}H)bremazocine in the presence of {mu} and {delta} blockers, selective for {kappa} binding sites. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the {mu}s site did not afford protection. These studies have demonstrated that when a disulfide bond at or near {mu} opioid binding sites was reduced, BAM could then alkylate this site, resulting in the specific irreversible labeling of {mu} opioid receptors.« less

Dynamic Expression of FKBP5 in the Medial Prefrontal Cortex Regulates Resiliency to Conditioned Fear

ERIC Educational Resources Information Center

Criado-Marrero, Marangelie; Morales Silva, Roberto J.; Velazquez, Bethzaly; Hernández, Anixa; Colon, María; Cruz, Emmanuel; Soler-Cedeño, Omar; Porter, James T.

2017-01-01

The factors influencing resiliency to the development of post-traumatic stress disorder (PTSD) remain to be elucidated. Clinical studies associate PTSD with polymorphisms of the FK506 binding protein 5 (FKBP5). However, it is unclear whether changes in FKBP5 expression alone could produce resiliency or susceptibility to PTSD-like symptoms. In this…

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

PubMed Central

Gumucio, D L; Rood, K L; Gray, T A; Riordan, M F; Sartor, C I; Collins, F S

1988-01-01

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation. Images PMID:2468996

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

PubMed

Schuller, Marion; Höfner, Georg; Wanner, Klaus T

2017-10-09

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Functional Proteomics to Identify Moderators of CD8+ T Cell Function in Melanoma

DTIC Science & Technology

2015-05-01

identified 17 phage that selectively bind TIL rather than effector cells. However, none of these phage influenced CD8+ TIL expansion or function in vitro...Using a novel NextGeneration sequencing approach, we have further defined another 1,000,000 phage that selectively bind TIL , of which 100,000 are unique...Using the original approach outlined in the application, we identified a total of 17 unique phage that selectively bind CD8+ TIL but not effector or

Biologically active ligands for yersinia outer protein H (YopH): feature based pharmacophore screening, docking and molecular dynamics studies.

PubMed

Tamilvanan, Thangaraju; Hopper, Waheeta

2014-01-01

Yersinia pestis, a Gram negative bacillus, spreads via lymphatic to lymph nodes and to all organs through the bloodstream, causing plague. Yersinia outer protein H (YopH) is one of the important effector proteins, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. The purpose of the study is to generate a three-dimensional (3D) pharmacophore model by using diverse sets of YopH inhibitors, which would be useful for designing of potential antitoxin. In this study, we have selected 60 biologically active inhibitors of YopH to perform Ligand based pharmacophore study to elucidate the important structural features responsible for biological activity. Pharmacophore model demonstrated the importance of two acceptors, one hydrophobic and two aromatic features toward the biological activity. Based on these features, different databases were screened to identify novel compounds and these ligands were subjected for docking, ADME properties and Binding energy prediction. Post docking validation was performed using molecular dynamics simulation for selected ligands to calculate the Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF). The ligands, ASN03270114, Mol_252138, Mol_31073 and ZINC04237078 may act as inhibitors against YopH of Y. pestis.

A Polymorphic p53 Response Element in KIT Ligand Influences Cancer Risk and Has Undergone Natural Selection

PubMed Central

Zeron-Medina, Jorge; Wang, Xuting; Repapi, Emmanouela; Campbell, Michelle R.; Su, Dan; Castro-Giner, Francesc; Davies, Benjamin; Peterse, Elisabeth F.P.; Sacilotto, Natalia; Walker, Graeme J.; Terzian, Tamara; Tomlinson, Ian P.; Box, Neil F.; Meinshausen, Nicolai; De Val, Sarah; Bell, Douglas A.; Bond, Gareth L.

2014-01-01

SUMMARY The ability of p53 to regulate transcription is crucial for tumor suppression and implies that inherited polymorphisms in functional p53-binding sites could influence cancer. Here, we identify a polymorphic p53 responsive element and demonstrate its influence on cancer risk using genome-wide data sets of cancer susceptibility loci, genetic variation, p53 occupancy, and p53-binding sites. We uncover a single-nucleotide polymorphism (SNP) in a functional p53-binding site and establish its influence on the ability of p53 to bind to and regulate transcription of the KITLG gene. The SNP resides in KITLG and associates with one of the largest risks identified among cancer genome-wide association studies. We establish that the SNP has undergone positive selection throughout evolution, signifying a selective benefit, but go on to show that similar SNPs are rare in the genome due to negative selection, indicating that polymorphisms in p53-binding sites are primarily detrimental to humans. PMID:24120139

Studies of the mechanism of selectivity of protein tyrosine phosphatase 1B (PTP1B) bidentate inhibitors using molecular dynamics simulations and free energy calculations.

PubMed

Fang, Lei; Zhang, Huai; Cui, Wei; Ji, Mingjun

2008-10-01

Bidentate inhibitors of protein tyrosine phosphatase 1B (PTP1B) are considered as a group of ideal inhibitors with high binding potential and high selectivity in treating type II diabetes. In this paper, the binding models of five bidentate inhibitors to PTP1B, TCPTP, and SHP-2 were investigated and compared by using molecular dynamics (MD) simulations and free energy calculations. The binding free energies were computed using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology. The calculation results show that the predicted free energies of the complexes are well consistent with the experimental data. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicates that the residues ARG24, ARG254, and GLN262 in the second binding site of PTP1B are essential for the high selectivity of inhibitors. Furthermore, the residue PHE182 close to the active site is also important for the selectivity and the binding affinity of the inhibitors. According to our analysis, it can be concluded that in most cases the polarity of the portion of the inhibitor that binds to the second binding site of the protein is positive to the affinity of the inhibitors while negative to the selectivity of the inhibitors. We expect that the information we obtained here can help to develop potential PTP1B inhibitors with more promising specificity.

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

PubMed Central

Deng, Gejing; Shen, Junqing; Yin, Ming; McManus, Jessica; Mathieu, Magali; Gee, Patricia; He, Timothy; Shi, Chaomei; Bedel, Olivier; McLean, Larry R.; Le-Strat, Frank; Zhang, Ying; Marquette, Jean-Pierre; Gao, Qiang; Zhang, Bailin; Rak, Alexey; Hoffmann, Dietmar; Rooney, Eamonn; Vassort, Aurelie; Englaro, Walter; Li, Yi; Patel, Vinod; Adrian, Francisco; Gross, Stefan; Wiederschain, Dmitri; Cheng, Hong; Licht, Stuart

2015-01-01

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg2+. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme. PMID:25391653

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

2018-05-03

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Ultrahigh and Selective SO2 Uptake in Inorganic Anion-Pillared Hybrid Porous Materials.

PubMed

Cui, Xili; Yang, Qiwei; Yang, Lifeng; Krishna, Rajamani; Zhang, Zhiguo; Bao, Zongbi; Wu, Hui; Ren, Qilong; Zhou, Wei; Chen, Banglin; Xing, Huabin

2017-07-01

The efficient capture of SO 2 is of great significance in gas-purification processes including flue-gas desulfurization and natural-gas purification, but the design of porous materials with high adsorption capacity and selectivity of SO 2 remains very challenging. Herein, the selective recognition and dense packing of SO 2 clusters through multiple synergistic host-guest and guest-guest interactions by controlling the pore chemistry and size in inorganic anion (SiF 6 2- , SIFSIX) pillared metal-organic frameworks is reported. The binding sites of anions and aromatic rings in SIFSIX materials grasp every atom of SO 2 firmly via S δ+ ···F δ- electrostatic interactions and O δ- ···H δ+ dipole-dipole interactions, while the guest-guest interactions between SO 2 molecules further promote gas trapping within the pore space, which is elucidated by first-principles density functional theory calculations and powder X-ray diffraction experiments. These interactions afford new benchmarks for the highly efficient removal of SO 2 from other gases, even if at a very low SO 2 concentration. Exceptionally high SO 2 capacity of 11.01 mmol g -1 is achieved at atmosphere pressure by SIFSIX-1-Cu, and unprecedented low-pressure SO 2 capacity is obtained in SIFSIX-2-Cu-i (4.16 mmol g -1 SO 2 at 0.01 bar and 2.31 mmol g -1 at 0.002 bar). More importantly, record SO 2 /CO 2 selectivity (86-89) and excellent SO 2 /N 2 selectivity (1285-3145) are also achieved. Experimental breakthrough curves further demonstrate the excellent performance of these hybrid porous materials in removing low-concentration SO 2 . © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway.

PubMed

Liu, Kuang-Kai; Qiu, Wei-Ru; Naveen Raj, Emmanuel; Liu, Huei-Fang; Huang, Hou-Syun; Lin, Yu-Wei; Chang, Chien-Jen; Chen, Ting-Hua; Chen, Chinpiao; Chang, Huan-Cheng; Hwang, Jenn-Kang; Chao, Jui-I

2017-01-02

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.

DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues.

PubMed

Ma, Xin; Guo, Jing; Sun, Xiao

2016-01-01

DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/.

Binding, Thermodynamics, and Selectivity of a Non-peptide Antagonist to the Melanocortin-4 Receptor

PubMed Central

Saleh, Noureldin; Kleinau, Gunnar; Heyder, Nicolas; Clark, Timothy; Hildebrand, Peter W.; Scheerer, Patrick

2018-01-01

The melanocortin-4 receptor (MC4R) is a potential drug target for treatment of obesity, anxiety, depression, and sexual dysfunction. Crystal structures for MC4R are not yet available, which has hindered successful structure-based drug design. Using microsecond-scale molecular-dynamics simulations, we have investigated selective binding of the non-peptide antagonist MCL0129 to a homology model of human MC4R (hMC4R). This approach revealed that, at the end of a multi-step binding process, MCL0129 spontaneously adopts a binding mode in which it blocks the agonistic-binding site. This binding mode was confirmed in subsequent metadynamics simulations, which gave an affinity for human hMC4R that matches the experimentally determined value. Extending our simulations of MCL0129 binding to hMC1R and hMC3R, we find that receptor subtype selectivity for hMC4R depends on few amino acids located in various structural elements of the receptor. These insights may support rational drug design targeting the melanocortin systems.

Evidence for estrogen receptor beta-selective activity of Vitex agnus-castus and isolated flavones.

PubMed

Jarry, Hubertus; Spengler, Barbara; Porzel, Andrea; Schmidt, Juergen; Wuttke, Wolfgang; Christoffel, Volker

2003-10-01

Recent cell culture experiments indicated that extracts of Vitex agnus-castus (VAC) may contain yet unidentified phytoestrogens. Estrogenic actions are mediated via estrogen receptors (ER). To investigate whether VAC compounds bind to the currently known isoforms ERalpha or ERss, ligand binding assays (LBA) were performed. Subtype specific ER-LBA revealed a binding of VAC to ERss only. To isolate the ERss-selective compounds, the extract was fractionated by bio-guidance. The flavonoid apigenin was isolated and identified as the most active ERss-selective phytoestrogen in VAC. Other isolated compounds were vitexin and penduletin. These data demonstrate that the phytoestrogens in VAC are ERss-selective.

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics

PubMed Central

2015-01-01

The crystal structures of channels and transporters reveal the chemical nature of ion-binding sites and, thereby, constrain mechanistic models for their transport processes. However, these structures, in and of themselves, do not reveal equilibrium selectivity or transport preferences, which can be discerned only from various functional assays. In this Review, I explore the relationship between cation transport protein structures, equilibrium binding measurements, and ion transport selectivity. The primary focus is on K+-selective channels and nonselective cation channels because they have been extensively studied both functionally and structurally, but the principles discussed are relevant to other transport proteins and molecules. PMID:26078056

Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors

PubMed Central

Del Valle-Mojica, Lisa M.; Cordero-Hernández, José M.; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A.; Ortíz, José G.

2011-01-01

The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [3H]Glutamate ([3H]Glu) and [3H]Fluorowillardine ([3H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38–60% of [3H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [3H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [3H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions. PMID:21151614

Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors.

PubMed

Del Valle-Mojica, Lisa M; Cordero-Hernández, José M; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A; Ortíz, José G

2011-01-01

The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [(3)H]Glutamate ([(3)H]Glu) and [(3)H]Fluorowillardine ([(3)H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38-60% of [(3)H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [(3)H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [(3)H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions.

Novel aspects of sialoglycan recognition by the Siglec-like domains of streptococcal SRR glycoproteins.

PubMed

Bensing, Barbara A; Khedri, Zahra; Deng, Lingquan; Yu, Hai; Prakobphol, Akraporn; Fisher, Susan J; Chen, Xi; Iverson, Tina M; Varki, Ajit; Sullam, Paul M

2016-11-01

Serine-rich repeat glycoproteins are adhesins expressed by commensal and pathogenic Gram-positive bacteria. A subset of these adhesins, expressed by oral streptococci, binds sialylated glycans decorating human salivary mucin MG2/MUC7, and platelet glycoprotein GPIb. Specific sialoglycan targets were previously identified for the ligand-binding regions (BRs) of GspB and Hsa, two serine-rich repeat glycoproteins expressed by Streptococcus gordonii While GspB selectively binds sialyl-T antigen, Hsa displays broader specificity. Here we examine the binding properties of four additional BRs from Streptococcus sanguinis or Streptococcus mitis and characterize the molecular determinants of ligand selectivity and affinity. Each BR has two domains that are essential for sialoglycan binding by GspB. One domain is structurally similar to the glycan-binding module of mammalian Siglecs (sialic acid-binding immunoglobulin-like lectins), including an arginine residue that is critical for glycan recognition, and that resides within a novel, conserved YTRY motif. Despite low sequence similarity to GspB, one of the BRs selectively binds sialyl-T antigen. Although the other three BRs are highly similar to Hsa, each displayed a unique ligand repertoire, including differential recognition of sialyl Lewis antigens and sulfated glycans. These differences in glycan selectivity were closely associated with differential binding to salivary and platelet glycoproteins. Specificity of sialoglycan adherence is likely an evolving trait that may influence the propensity of streptococci expressing Siglec-like adhesins to cause infective endocarditis. Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Binding site exploration of CCR5 using in silico methodologies: a 3D-QSAR approach.

PubMed

Gadhe, Changdev G; Kothandan, Gugan; Cho, Seung Joo

2013-01-01

Chemokine receptor 5 (CCR5) is an important receptor used by human immunodeficiency virus type 1 (HIV-1) to gain viral entry into host cell. In this study, we used a combined approach of comparative modeling, molecular docking, and three dimensional quantitative structure activity relationship (3D-QSAR) analyses to elucidate detailed interaction of CCR5 with their inhibitors. Docking study of the most potent inhibitor from a series of compounds was done to derive the bioactive conformation. Parameters such as random selection, rational selection, different charges and grid spacing were utilized in the model development to check their performance on the model predictivity. Final comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models were chosen based on the rational selection method, Gasteiger-Hückel charges and a grid spacing of 0.5 Å. Rational model for CoMFA (q(2) = 0.722, r(2) = 0.884, Q(2) = 0.669) and CoMSIA (q(2) = 0.712, r(2) = 0.825, Q(2) = 0.522) was obtained with good statistics. Mapping of contour maps onto CCR5 interface led us to better understand of the ligand-protein interaction. Docking analysis revealed that the Glu283 is crucial for interaction. Two new amino acid residues, Tyr89 and Thr167 were identified as important in ligand-protein interaction. No site directed mutagenesis studies on these residues have been reported.

Electrophysiological characterization of 14-benzoyltalatisamine, a selective blocker of the delayed rectifier K+ channel found in virtual screening.

PubMed

Song, Ming-Ke; Liu, Hong; Jiang, Hua-Liang; Yue, Jian-Min; Hu, Guo-Yuan

2006-02-15

14-Benzoyltalatisamine is a potent and selective blocker of the delayed rectifier K+ channel found in a computational virtual screening study. The compound was found to block the K+ channel from the extracellular side. However, it is unclear whether 14-benzoyltalatisamine shares the same block mechanism with tetraethylammonium (TEA). In order to elucidate how the hit compound found by the virtual screening interacts with the outer vestibule of the K+ channel, the effects of 14-benzoyltalatisamine and TEA on the delayed rectifier K+ current of rat dissociated hippocampal neurons were compared using whole-cell voltage-clamp recording. External application of 14-benzoyltalatisamine and TEA reversibly inhibited the current with IC50 values of 10.1+/-2.2 microM and 1.05+/-0.21 mM, respectively. 14-Benzoyltalatisamine exerted voltage-dependent inhibition, markedly accelerated the decay of the current, and caused a significant hyperpolarizing shift of the steady-state activation curve, whereas TEA caused voltage-independent inhibition, without affecting the kinetic parameters of the current. The blockade by 14-benzoyltalatisamine, but not by TEA, was significantly diminished in a high K+ (60 mM) external solution. The potency of 14-benzoyltalatisamine was markedly reduced in the presence of 15 mM TEA. The results suggest that 14-benzoyltalatisamine bind to the external pore entry of the delayed rectifier K+ channel with partial insertion into the selectivity filter, which is in conformity with that predicted by the molecular docking model in the virtual screening.

Aerobic sn-glycerol-3-phosphate dehydrogenase from Escherichia coli binds to the cytoplasmic membrane through an amphipathic alpha-helix.

PubMed Central

Walz, Antje-Christine; Demel, Rudy A; de Kruijff, Ben; Mutzel, Rupert

2002-01-01

sn-Glycerol-3-phosphate dehydrogenase (GlpD) from Escherichia coli is a peripheral membrane enzyme involved in respiratory electron transfer. For it to display its enzymic activity, binding to the inner membrane is required. The way the enzyme interacts with the membrane and how this controls activity has not been elucidated. In the present study we provide evidence for direct protein-lipid interaction. Using the monolayer technique, we observed insertion of GlpD into lipid monolayers with a clear preference for anionic phospholipids. GlpD variants with point mutations in their predicted amphipathic helices showed a decreased ability to penetrate anionic phospholipid monolayers. From these data we propose that membrane binding of GlpD occurs by insertion of an amphipathic helix into the acyl-chain region of lipids mediated by negatively charged phospholipids. PMID:11955283

Towards novel therapeutics for HIV through fragment-based screening and drug design.

PubMed

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Truncated ALK derived from chromosomal translocation t(2;5)(p23;q35) binds to the SH3 domain of p85-PI3K.

PubMed

Polgar, Doris; Leisser, Christina; Maier, Susanne; Strasser, Stephan; Rüger, Beate; Dettke, Markus; Khorchide, Maya; Simonitsch, Ingrid; Cerni, Christa; Krupitza, Georg

2005-02-15

The chromosomal translocation t(2;5)(p23;q35) is associated with "Anaplastic large cell lymphomas" (ALCL), a Non Hodgkin Lymphoma occurring in childhood. The fusion of the tyrosine kinase gene-ALK (anaplastic lymphoma kinase) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35 results in a 80 kDa chimeric protein, which activates the "survival" kinase PI3K. However, the binding mechanism between truncated ALK and PI3K is poorly understood. Therefore, we attempted to elucidate the molecular interaction between ALK and the regulatory p85 subunit of PI3K. Here we provide evidence that the truncated ALK homodimer binds to the SH3 domain of p85. This finding may be useful for the development of a new target-specific intervention.

Reverse engineering the cooperative machinery of human hemoglobin.

PubMed

Ren, Zhong

2013-01-01

Hemoglobin transports molecular oxygen from the lungs to all human tissues for cellular respiration. Its α2β2 tetrameric assembly undergoes cooperative binding and releasing of oxygen for superior efficiency and responsiveness. Over past decades, hundreds of hemoglobin structures were determined under a wide range of conditions for investigation of molecular mechanism of cooperativity. Based on a joint analysis of hemoglobin structures in the Protein Data Bank (Ren, companion article), here I present a reverse engineering approach to elucidate how two subunits within each dimer reciprocate identical motions that achieves intradimer cooperativity, how ligand-induced structural signals from two subunits are integrated to drive quaternary rotation, and how the structural environment at the oxygen binding sites alter their binding affinity. This mechanical model reveals the intricate design that achieves the cooperative mechanism and has previously been masked by inconsistent structural fluctuations. A number of competing theories on hemoglobin cooperativity and broader protein allostery are reconciled and unified.

Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells.

PubMed

La, Xiaoqin; Zhang, Lichao; Li, Zhuoyu; Yang, Peng; Wang, Yingying

2017-03-28

Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34.

Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

PubMed Central

Li, Zhuoyu; Yang, Peng; Wang, Yingying

2017-01-01

Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34. PMID:28157699

Synthesis and crystal structure determination of copper(II)-complex: In vitro DNA and HSA binding, pBR322 plasmid cleavage, cell imaging and cytotoxic studies.

PubMed

Tabassum, Sartaj; Zaki, Mehvash; Ahmad, Musheer; Afzal, Mohd; Srivastav, Saurabh; Srikrishna, Saripella; Arjmand, Farukh

2014-08-18

New Cu(II) complex 1 of indole-3-propionic acid and 1,10-phenanthroline was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. In vitro DNA binding studies of 1 was performed by employing UV-vis and fluorescence spectroscopic techniques. The binding affinity towards human serum albumin (HSA) was also investigated to understand the carrier role in body system, as the time dependent HPLC experiment of 1 revealed that bonded drug with protein releases slowly in presence of DNA. Complex 1 exhibited good anti-tumor activity (GI50 values <10 μg/ml), and to elucidate the mechanism of tumor inhibition, topoisomerase I enzymatic activity was carried out and further validated by cell imaging studies which clearly showed its nuclear localization. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

CJ-1639: A Potent and Highly Selective Dopamine D3 Receptor Full Agonist.

PubMed

Chen, Jianyong; Collins, Gregory T; Levant, Beth; Woods, James; Deschamps, Jeffrey R; Wang, Shaomeng

2011-08-11

We have identified several ligands with high binding affinities to the dopamine D3 receptor and excellent selectivity over the D2 and D1 receptors. CJ-1639 (17) binds to the D3 receptor with a K(i) value of 0.50 nM and displays a selectivity of >5,000 times over D2 and D1 receptors in binding assays using dopamine receptors expressed in the native rat brain tissues. CJ-1639 binds to human D3 receptor with a K(i) value of 3.61 nM and displays over >1000-fold selectivity over human D1 and D2 receptors. CJ-1639 is active at 0.01 mg/kg at the dopamine D3 receptor in the rat and only starts to show a modest D2 activity at doses as high as 10 mg/kg. CJ-1639 is the most potent and selective D3 full agonist reported to date.

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

PubMed

Kandeel, Mahmoud; Kitade, Yukio

2018-02-01

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid, Optimized Interactomic Screening

PubMed Central

Hakhverdyan, Zhanna; Domanski, Michal; Hough, Loren; Oroskar, Asha A.; Oroskar, Anil R.; Keegan, Sarah; Dilworth, David J.; Molloy, Kelly R.; Sherman, Vadim; Aitchison, John D.; Fenyö, David; Chait, Brian T.; Jensen, Torben Heick; Rout, Michael P.; LaCava, John

2015-01-01

We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes. PMID:25938370

Principal component analysis of chemical shift perturbation data of a multiple-ligand-binding system for elucidation of respective binding mechanism.

PubMed

Konuma, Tsuyoshi; Lee, Young-Ho; Goto, Yuji; Sakurai, Kazumasa

2013-01-01

Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple-ligand-binding system, determining quantitative parameters such as a dissociation constant (K(d) ) is difficult. Here, we used a method we named CS-PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine β-lactoglobulin (βLG) and 1-anilinonaphthalene-8-sulfonate (ANS), which is a multiple-ligand-binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of βLG-ANS complexes for each binding site. In addition, we determined the K(d) values as 3.42 × 10⁻⁴ M² and 2.51 × 10⁻³ M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable K(d) values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the βLG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS-PCA was confirmed to provide not only the positions and the K(d) values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate protein-ligand interactions. Copyright © 2012 Wiley Periodicals, Inc.

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

PubMed

Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

2008-11-21

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

Microbial Ecology of Thermophilic Anaerobic Digestion. Final Report

DOE R&D Accomplishments Database

Zinder, Stephen H.

2000-04-15

This grant supported research on methanogenic archaea. The two major areas that were supported were conversion of acetic acid to methane and nitrogen fixation by Methanosarcina. Among the achievements of this research were the isolation of novel methanogenic cultures, elucidation of the pathways from acetate to methane, description of a specific DNA-binding complex in nitrogen fixing methanogens, and demonstration of an alternative nitrogenase in Methanosarcina.

Structural insight into catalytic mechanism of PET hydrolase.

PubMed

Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

2017-12-13

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.