Tris-amidoximate uranyl complexes via η2 binding mode coordinated in aqueous solution shown by X-ray absorption spectroscopy and density functional theory methods.

PubMed

Zhang, Linjuan; Qie, Meiying; Su, Jing; Zhang, Shuo; Zhou, Jing; Li, Jiong; Wang, Yu; Yang, Shitong; Wang, Shuao; Li, Jingye; Wu, Guozhong; Wang, Jian Qiang

2018-03-01

The present study sheds some light on the long-standing debate concerning the coordination properties between uranyl ions and the amidoxime ligand, which is a key ingredient for achieving efficient extraction of uranium. Using X-ray absorption fine structure combined with theoretical simulation methods, the binding mode and bonding nature of a uranyl-amidoxime complex in aqueous solution were determined for the first time. The results show that in a highly concentrated amidoxime solution the preferred binding mode between UO 2 2+ and the amidoxime ligand is η 2 coordination with tris-amidoximate species. In such a uranyl-amidoximate complex with η 2 binding motif, strong covalent interaction and orbital hybridization between U 5f/6d and (N, O) 2p should be responsible for the excellent binding ability of the amidoximate ligand to uranyl. The study was performed directly in aqueous solution to avoid the possible binding mode differences caused by crystallization of a single-crystal sample. This work also is an example of the simultaneous study of local structure and electronic structure in solution systems using combined diagnostic tools.

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.

Accuracy of binding mode prediction with a cascadic stochastic tunneling method.

PubMed

Fischer, Bernhard; Basili, Serena; Merlitz, Holger; Wenzel, Wolfgang

2007-07-01

We investigate the accuracy of the binding modes predicted for 83 complexes of the high-resolution subset of the ASTEX/CCDC receptor-ligand database using the atomistic FlexScreen approach with a simple forcefield-based scoring function. The median RMS deviation between experimental and predicted binding mode was just 0.83 A. Over 80% of the ligands dock within 2 A of the experimental binding mode, for 60 complexes the docking protocol locates the correct binding mode in all of ten independent simulations. Most docking failures arise because (a) the experimental structure clashed in our forcefield and is thus unattainable in the docking process or (b) because the ligand is stabilized by crystal water. 2007 Wiley-Liss, Inc.

Automated large-scale file preparation, docking, and scoring: evaluation of ITScore and STScore using the 2012 Community Structure-Activity Resource benchmark.

PubMed

Grinter, Sam Z; Yan, Chengfei; Huang, Sheng-You; Jiang, Lin; Zou, Xiaoqin

2013-08-26

In this study, we use the recently released 2012 Community Structure-Activity Resource (CSAR) data set to evaluate two knowledge-based scoring functions, ITScore and STScore, and a simple force-field-based potential (VDWScore). The CSAR data set contains 757 compounds, most with known affinities, and 57 crystal structures. With the help of the script files for docking preparation, we use the full CSAR data set to evaluate the performances of the scoring functions on binding affinity prediction and active/inactive compound discrimination. The CSAR subset that includes crystal structures is used as well, to evaluate the performances of the scoring functions on binding mode and affinity predictions. Within this structure subset, we investigate the importance of accurate ligand and protein conformational sampling and find that the binding affinity predictions are less sensitive to non-native ligand and protein conformations than the binding mode predictions. We also find the full CSAR data set to be more challenging in making binding mode predictions than the subset with structures. The script files used for preparing the CSAR data set for docking, including scripts for canonicalization of the ligand atoms, are offered freely to the academic community.

Distinct mechanisms of recognizing endosomal sorting complex required for transport III (ESCRT-III) protein IST1 by different microtubule interacting and trafficking (MIT) domains.

PubMed

Guo, Emily Z; Xu, Zhaohui

2015-03-27

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

DOE PAGES

Guo, Emily Z.; Xu, Zhaohui

2015-02-05

The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). In this paper, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed thatmore » IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. Finally, these observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode.« less

Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt Dependent Thermodynamics

PubMed Central

Koh, Junseock; Shkel, Irina; Saecker, Ruth M.; Record, M. Thomas

2011-01-01

Previous ITC and FRET studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34 bp mode which interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it 140° and thereby increasing its flexibility, and two weaker, modestly cooperative small-site-size modes (10 bp, 6 bp) useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and deduce that DNA in the 34 bp mode is bent around but not wrapped on the body of HU, in contrast to specific binding of IHF. Analyses of binding isotherms (8, 15, 34 bp DNA) and initial binding heats (34, 38, 160 bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; most probable values of Ski on 34 bp or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent-DNA 34 bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6 bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed. PMID:21513716

Predicting binding modes of reversible peptide-based inhibitors of falcipain-2 consistent with structure-activity relationships.

PubMed

Hernández González, Jorge Enrique; Hernández Alvarez, Lilian; Pascutti, Pedro Geraldo; Valiente, Pedro A

2017-09-01

Falcipain-2 (FP-2) is a major hemoglobinase of Plasmodium falciparum, considered an important drug target for the development of antimalarials. A previous study reported a novel series of 20 reversible peptide-based inhibitors of FP-2. However, the lack of tridimensional structures of the complexes hinders further optimization strategies to enhance the inhibitory activity of the compounds. Here we report the prediction of the binding modes of the aforementioned inhibitors to FP-2. A computational approach combining previous knowledge on the determinants of binding to the enzyme, docking, and postdocking refinement steps, is employed. The latter steps comprise molecular dynamics simulations and free energy calculations. Remarkably, this approach leads to the identification of near-native ligand conformations when applied to a validation set of protein-ligand structures. Overall, we proposed substrate-like binding modes of the studied compounds fulfilling the structural requirements for FP-2 binding and yielding free energy values that correlated well with the experimental data. Proteins 2017; 85:1666-1683. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The binding modes of carbazole derivatives with telomere G-quadruplex

NASA Astrophysics Data System (ADS)

Zhang, Xiu-feng; Zhang, Hui-juan; Xiang, Jun-feng; Li, Qian; Yang, Qian-fan; Shang, Qian; Zhang, Yan-xia; Tang, Ya-lin

2010-10-01

It is reported that carbazole derivatives can stabilize G-quadruplex DNA structure formed by human telomeric sequence, and therefore, they have the potential to serve as anti-cancer agents. In this present study, in order to further explore the binding mode between carbazole derivatives and G-quadruplex formed by human telomeric sequence, two carbazole iodides (BMVEC, MVEC) molecules were synthesized and used to investigate the interaction with the human telomeric parallel and antiparallel G-quadruplex structures by NMR, CD and molecular modeling study. Interestingly, it is the pivotal the cationic charge pendant groups of pyridinium rings of carbazole that plays an essential role in the stabilizing and binding mode of the human telomeric sequences G-quadruplex structure. It was found that BMVEC with two cationic charge pendant groups of pyridinium rings of 9-ethylcarbazole cannot only stabilize parallel G-quadruple of Hum6 by groove binding and G-tetrad stacking modes and antiparallel G-quadruplex of Hum22 by groove binding, but also induce the formation of mixed G-quadruplex of Hum22. While MVEC with one cationic charge pendant groups of pyridinium ring only can bind with the parallel G-quadruplex of Hum6 by the stacking onto the G4 G-tetrad and could not interact with the G-quadruplex of Hum22.

Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family.

PubMed

Contessa, Gian Marco; Orsale, Maria; Melino, Sonia; Torre, Vincent; Paci, Maurizio; Desideri, Alessandro; Cicero, Daniel O

2005-03-01

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

Pyrrole-Based Antitubulin Agents: Two Distinct Binding Modalities are Predicted for C-2 Analogs in the Colchicine Site.

PubMed

Da, Chenxiao; Telang, Nakul; Barelli, Peter; Jia, Xin; Gupton, John T; Mooberry, Susan L; Kellogg, Glen E

2012-01-12

3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester is a promising antitubulin lead agent that targets the colchicine site of tubulin. C-2 analogs were synthesized and tested for microtubule depolymerizing and antiproliferative activity. Molecular modeling studies using both GOLD docking and HINT (Hydropathic INTeraction) scoring revealed two distinct binding modes that explain the structural-activity relationships and are in accord with the structural basis of colchicine binding to tubulin. The binding mode of higher activity compounds is buried deeper in the site and overlaps well with rings A and C of colchicine, while the lower activity binding mode shows fewer critical contacts with tubulin. The model distinguishes highly active compounds from those with weaker activities and provides novel insights into the colchicine site and compound design.

A computational analysis of the binding mode of closantel as inhibitor of the Onchocerca volvulus chitinase: insights on macrofilaricidal drug design

NASA Astrophysics Data System (ADS)

Segura-Cabrera, Aldo; Bocanegra-García, Virgilio; Lizarazo-Ortega, Cristian; Guo, Xianwu; Correa-Basurto, José; Rodríguez-Pérez, Mario A.

2011-12-01

Onchocerciasis is a leading cause of blindness with at least 37 million people infected and more than 120 million people at risk of contracting the disease; most (99%) of this population, threatened by infection, live in Africa. The drug of choice for mass treatment is the microfilaricidal Mectizan® (ivermectin); it does not kill the adult stages of the parasite at the standard dose which is a single annual dose aimed at disease control. However, multiple treatments a year with ivermectin have effects on adult worms. The discovery of new therapeutic targets and drugs directed towards the killing of the adult parasites are thus urgently needed. The chitinase of filarial nematodes is a new drug target due to its essential function in the metabolism and molting of the parasite. Closantel is a potent and specific inhibitor of chitinase of Onchocerca volvulus (OvCHT1) and other filarial chitinases. However, the binding mode and specificity of closantel towards OvCHT1 remain unknown. In the absence of a crystallographic structure of OvCHT1, we developed a homology model of OvCHT1 using the currently available X-ray structures of human chitinases as templates. Energy minimization and molecular dynamics (MD) simulation of the model led to a high quality of 3D structure of OvCHIT1. A flexible docking study using closantel as the ligand on the binding site of OvCHIT1 and human chitinases was performed and demonstrated the differences in the closantel binding mode between OvCHIT1 and human chitinase. Furthermore, molecular dynamics simulations and free-energy calculation were employed to determine and compare the detailed binding mode of closantel with OvCHT1 and the structure of human chitinase. This comparative study allowed identification of structural features and properties responsible for differences in the computationally predicted closantel binding modes. The homology model and the closantel binding mode reported herein might help guide the rational development of novel drugs against the adult parasite of O. volvulus and such findings could be extrapolated to other filarial neglected diseases.

Computational determination of the binding mode of α-conotoxin to nicotinic acetylcholine receptor

NASA Astrophysics Data System (ADS)

Tabassum, Nargis; Yu, Rilei; Jiang, Tao

2016-12-01

Conotoxins belong to the large families of disulfide-rich peptide toxins from cone snail venom, and can act on a broad spectrum of ion channels and receptors. They are classified into different subtypes based on their targets. The α-conotoxins selectively inhibit the current of the nicotinic acetylcholine receptors. Because of their unique selectivity towards distinct nAChR subtypes, α-conotoxins become valuable tools in nAChR study. In addition to the X-ray structures of α-conotoxins in complex with acetylcholine-binding protein, a homolog of the nAChR ligand-binding domain, the high-resolution crystal structures of the extracellular domain of the α1 and α9 subunits are also obtained. Such structures not only revealed the details of the configuration of nAChR, but also provided higher sequence identity templates for modeling the binding modes of α-conotoxins to nAChR. This mini-review summarizes recent modeling studies for the determination of the binding modes of α-conotoxins to nAChR. As there are not crystal structures of the nAChR in complex with conotoxins, computational modeling in combination of mutagenesis data is expected to reveal the molecular recognition mechanisms that govern the interactions between α-conotoxins and nAChR at molecular level. An accurate determination of the binding modes of α-conotoxins on AChRs allows rational design of α-conotoxin analogues with improved potency or selectivity to nAChRs.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed

Iversen, L F; Brzozowski, M; Hastrup, S; Hubbard, R; Kastrup, J S; Larsen, I K; Naerum, L; Nørskov-Lauridsen, L; Rasmussen, P B; Thim, L; Wiberg, F C; Lundgren, K

1997-05-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed Central

Iversen, L. F.; Brzozowski, M.; Hastrup, S.; Hubbard, R.; Kastrup, J. S.; Larsen, I. K.; Naerum, L.; Nørskov-Lauridsen, L.; Rasmussen, P. B.; Thim, L.; Wiberg, F. C.; Lundgren, K.

1997-01-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand. PMID:9144768

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

PubMed

Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

2018-06-16

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

PubMed Central

Hinz, Andreas; Lutje Hulsik, David; Forsman, Anna; Koh, Willie Wee-Lee; Belrhali, Hassan; Gorlani, Andrea; de Haard, Hans; Weiss, Robin A.; Verrips, Theo; Weissenhorn, Winfried

2010-01-01

HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site. PMID:20463957

Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

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

Ida, Tomoyo; Suzuki, Hideyuki; Fukuyama, Keiichi

2014-02-01

The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalentlymore » through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.« less

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.

How Does Mg2+ Modulate the RNA Folding Mechanism: A Case Study of the G:C W:W Trans Basepair.

PubMed

Halder, Antarip; Roy, Rohit; Bhattacharyya, Dhananjay; Mitra, Abhijit

2017-07-25

Reverse Watson-Crick G:C basepairs (G:C W:W Trans) occur frequently in different functional RNAs. This is one of the few basepairs whose gas-phase-optimized isolated geometry is inconsistent with the corresponding experimental geometry. Several earlier studies indicate that through post-transcriptional modification, direct protonation, or coordination with Mg 2+ , accumulation of positive charge near N7 of guanine can stabilize the experimental geometry. Interestingly, recent studies reveal significant variation in the position of putatively bound Mg 2+ . This, in conjunction with recently raised doubts regarding some of the Mg 2+ assignments near the imino nitrogen of guanine, is suggestive of the existence of multiple Mg 2+ binding modes for this basepair. Our detailed investigation of Mg 2+ -bound G:C W:W Trans pairs occurring in high-resolution RNA crystal structures shows that they are found in 14 different contexts, eight of which display Mg 2+ binding at the Hoogsteen edge of guanine. Further examination of occurrences in these eight contexts led to the characterization of three different Mg 2+ binding modes: 1) direct binding via N7 coordination, 2) direct binding via O6 coordination, and 3) binding via hydrogen-bonding interaction with the first-shell water molecules. In the crystal structures, the latter two modes are associated with a buckled and propeller-twisted geometry of the basepair. Interestingly, respective optimized geometries of these different Mg 2+ binding modes (optimized using six different DFT functionals) are consistent with their corresponding experimental geometries. Subsequent interaction energy calculations at the MP2 level, and decomposition of its components, suggest that for G:C W:W Trans , Mg 2+ binding can fine tune the basepair geometries without compromising with their stability. Our results, therefore, underline the importance of the mode of binding of Mg 2+ ions in shaping RNA structure, folding and function. Copyright © 2017. Published by Elsevier Inc.

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

NASA Astrophysics Data System (ADS)

Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

2017-08-01

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

Reassessment of the Unique Mode of Binding between Angiotensin II Type 1 Receptor and Their Blockers

PubMed Central

Matsuo, Yoshino; Saku, Keijiro; Karnik, Sadashiva S.

2013-01-01

While the molecular structures of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT1 receptor, different positions of the AT1 receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT1 receptor, Tyr113, Tyr184, Lys199, His256 and Gln257 using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT1 receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr113 in the AT1 receptor and the hydroxyl group of olmesartan, between Lys199 and carboxyl or tetrazole groups, and between His256 or Gln257 and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys199, His256 and Gln257 of AT1 receptor. Lys199 in the AT1 receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys199. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr113. On the other hand, the n-butyl group of irbesartan may bind to Tyr113. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT1 receptor and for the formation of unique modes of binding. PMID:24260317

Detailed Analysis of the Binding Mode of Vanilloids to Transient Receptor Potential Vanilloid Type I (TRPV1) by a Mutational and Computational Study

PubMed Central

Mori, Yoshikazu; Ogawa, Kazuo; Warabi, Eiji; Yamamoto, Masahiro; Hirokawa, Takatsugu

2016-01-01

Transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel and a multimodal sensor protein. Since the precise structure of TRPV1 was obtained by electron cryo-microscopy, the binding mode of representative agonists such as capsaicin and resiniferatoxin (RTX) has been extensively characterized; however, detailed information on the binding mode of other vanilloids remains lacking. In this study, mutational analysis of human TRPV1 was performed, and four agonists (capsaicin, RTX, [6]-shogaol and [6]-gingerol) were used to identify amino acid residues involved in ligand binding and/or modulation of proton sensitivity. The detailed binding mode of each ligand was then simulated by computational analysis. As a result, three amino acids (L518, F591 and L670) were newly identified as being involved in ligand binding and/or modulation of proton sensitivity. In addition, in silico docking simulation and a subsequent mutational study suggested that [6]-gingerol might bind to and activate TRPV1 in a unique manner. These results provide novel insights into the binding mode of various vanilloids to the channel and will be helpful in developing a TRPV1 modulator. PMID:27606946

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

PubMed Central

Heng, Jie; Zhao, Yan; Liu, Ming; Liu, Yue; Fan, Junping; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

2015-01-01

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters. PMID:26238402

Structural basis of redox-dependent substrate binding of protein disulfide isomerase

PubMed Central

Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

2015-01-01

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

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

NASA Astrophysics Data System (ADS)

Duan, Rui; Xu, Xianjin; Zou, Xiaoqin

2018-01-01

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

Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2.

PubMed

Xu, Emma-Ruoqi; Blythe, Emily E; Fischer, Gerhard; Hyvönen, Marko

2017-07-28

Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure-based Understanding of Binding Affinity and Mode ...

EPA Pesticide Factsheets

The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interactions and specific hydrogen bonds with the ligand. Here we present a framework for quantitative analysis of the steric and electronic features of the human ERα-ligand complex using three dimensional (3D) protein-ligand interaction description combined with 3D-QSAR approach. An empirical hydrophobicity density field is applied to account for hydrophobic contacts of ligand within the LBP. The obtained 3D-QSAR model revealed that hydrophobic contacts primarily determine binding affinity and govern binding mode with hydrogen bonds. Several residues of the LBP appear to be quite flexible and adopt a spectrum of conformations in various ERα-ligand complexes, in particular His524. The 3D-QSAR was combined with molecular docking based on three receptor conformations to accommodate receptor flexibility. The model indicates that the dynamic character of the LBP allows accommodation and stable binding of structurally diverse ligands, and proper representation of the protein flexibility is critical for reasonable description of binding of the ligands. Our results provide a quantitative and mechanistic understanding of binding affinity and mode of ERα agonists and antagonists that may be applicab

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

PubMed

Delaforge, Elise; Kragelj, Jaka; Tengo, Laura; Palencia, Andrés; Milles, Sigrid; Bouvignies, Guillaume; Salvi, Nicola; Blackledge, Martin; Jensen, Malene Ringkjøbing

2018-01-24

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Crystal structure of glucose isomerase in complex with xylitol inhibitor in one metal binding mode.

PubMed

Bae, Ji-Eun; Kim, In Jung; Nam, Ki Hyun

2017-11-04

Glucose isomerase (GI) is an intramolecular oxidoreductase that interconverts aldoses and ketoses. These characteristics are widely used in the food, detergent, and pharmaceutical industries. In order to obtain an efficient GI, identification of novel GI genes and substrate binding/inhibition have been studied. Xylitol is a well-known inhibitor of GI. In Streptomyces rubiginosus, two crystal structures have been reported for GI in complex with xylitol inhibitor. However, a structural comparison showed that xylitol can have variable conformation at the substrate binding site, e.g., a nonspecific binding mode. In this study, we report the crystal structure of S. rubiginosus GI in a complex with xylitol and glycerol. Our crystal structure showed one metal binding mode in GI, which we presumed to represent the inactive form of the GI. The metal ion was found only at the M1 site, which was involved in substrate binding, and was not present at the M2 site, which was involved in catalytic function. The O 2 and O 4 atoms of xylitol molecules contributed to the stable octahedral coordination of the metal in M1. Although there was no metal at the M2 site, no large conformational change was observed for the conserved residues coordinating M2. Our structural analysis showed that the metal at the M2 site was not important when a xylitol inhibitor was bound to the M1 site in GI. Thus, these findings provided important information for elucidation or engineering of GI functions. Copyright © 2017 Elsevier Inc. All rights reserved.

A flexible docking scheme to explore the binding selectivity of PDZ domains.

PubMed

Gerek, Z Nevin; Ozkan, S Banu

2010-05-01

Modeling of protein binding site flexibility in molecular docking is still a challenging problem due to the large conformational space that needs sampling. Here, we propose a flexible receptor docking scheme: A dihedral restrained replica exchange molecular dynamics (REMD), where we incorporate the normal modes obtained by the Elastic Network Model (ENM) as dihedral restraints to speed up the search towards correct binding site conformations. To our knowledge, this is the first approach that uses ENM modes to bias REMD simulations towards binding induced fluctuations in docking studies. In our docking scheme, we first obtain the deformed structures of the unbound protein as initial conformations by moving along the binding fluctuation mode, and perform REMD using the ENM modes as dihedral restraints. Then, we generate an ensemble of multiple receptor conformations (MRCs) by clustering the lowest replica trajectory. Using ROSETTALIGAND, we dock ligands to the clustered conformations to predict the binding pose and affinity. We apply this method to postsynaptic density-95/Dlg/ZO-1 (PDZ) domains; whose dynamics govern their binding specificity. Our approach produces the lowest energy bound complexes with an average ligand root mean square deviation of 0.36 A. We further test our method on (i) homologs and (ii) mutant structures of PDZ where mutations alter the binding selectivity. In both cases, our approach succeeds to predict the correct pose and the affinity of binding peptides. Overall, with this approach, we generate an ensemble of MRCs that leads to predict the binding poses and specificities of a protein complex accurately.

A flexible docking scheme to explore the binding selectivity of PDZ domains

PubMed Central

Gerek, Z Nevin; Ozkan, S Banu

2010-01-01

Modeling of protein binding site flexibility in molecular docking is still a challenging problem due to the large conformational space that needs sampling. Here, we propose a flexible receptor docking scheme: A dihedral restrained replica exchange molecular dynamics (REMD), where we incorporate the normal modes obtained by the Elastic Network Model (ENM) as dihedral restraints to speed up the search towards correct binding site conformations. To our knowledge, this is the first approach that uses ENM modes to bias REMD simulations towards binding induced fluctuations in docking studies. In our docking scheme, we first obtain the deformed structures of the unbound protein as initial conformations by moving along the binding fluctuation mode, and perform REMD using the ENM modes as dihedral restraints. Then, we generate an ensemble of multiple receptor conformations (MRCs) by clustering the lowest replica trajectory. Using RosettaLigand, we dock ligands to the clustered conformations to predict the binding pose and affinity. We apply this method to postsynaptic density-95/Dlg/ZO-1 (PDZ) domains; whose dynamics govern their binding specificity. Our approach produces the lowest energy bound complexes with an average ligand root mean square deviation of 0.36 Å. We further test our method on (i) homologs and (ii) mutant structures of PDZ where mutations alter the binding selectivity. In both cases, our approach succeeds to predict the correct pose and the affinity of binding peptides. Overall, with this approach, we generate an ensemble of MRCs that leads to predict the binding poses and specificities of a protein complex accurately. PMID:20196074

Insights into the Mutation-Induced HHH Syndrome from Modeling Human Mitochondrial Ornithine Transporter-1

PubMed Central

Wang, Jing-Fang; Chou, Kuo-Chen

2012-01-01

Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109–113). Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i) the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii) the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii) the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and developing effective drugs against the disease. PMID:22292090

Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

NASA Astrophysics Data System (ADS)

Vanderlinden, Willem; Lipfert, Jan; Demeulemeester, Jonas; Debyser, Zeger; de Feyter, Steven

2014-04-01

LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease.LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease. Electronic supplementary information (ESI) available: SFM topographs of phage lambda DNA in situ, in the absence and presence of LEDGF/p75; model-independent tests for DNA chain equilibration in 2D; SFM topographs of plasmid DNA substrates I-IV in the absence of LEDGF/p75; proof-of-principle of bend angle determination on supercoiled plasmid DNA-EcoRV binding to cognate and non-cognate sites in pBR322 plasmid DNA. See DOI: 10.1039/c4nr00022f

Homotropic Cooperativity from the Activation Pathway of the Allosteric Ligand-Responsive Regulatory Protein TRAP†

PubMed Central

Kleckner, Ian R.; McElroy, Craig A.; Kuzmic, Petr; Gollnick, Paul; Foster, Mark P.

2014-01-01

The trp RNA-binding Attenuation Protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both μs-ms rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of eleven bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and we observed well-separated kinetic steps. These data were analyzed using non-linear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that Trp dissociation from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation, and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery. PMID:24224873

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.

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.

DNA Binding Mode Transitions of Escherichia coli HUαβ: Evidence for Formation of a Bent DNA – Protein Complex on Intact, Linear Duplex DNA

PubMed Central

Koh, Junseock; Saecker, Ruth M.; Record, M. Thomas

2008-01-01

Escherichia coli HUαβ, a major nucleoid associated protein (NAP), organizes the DNA chromosome and facilitates numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA lengths (8, 15, 34, 38 and 160 base pairs) we establish that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA mole ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 base pairs), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 (± 0.4) × 106 M−1, and a binding enthalpy of +7.7 (± 0.6) kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω ~ 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 (± 0.2) × 105 M−1 for the 10 bp mode, 3.5 (± 1.4) × 104 M−1 for the 6 bp mode) and binding enthalpies (4.2 (± 0.3) kcal/mol for the 10 bp mode, −1.6 (±0.3) kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. FRET data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement, AFM), fluorescence, and electrophoretic gel mobility shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. PMID:18657548

Binding modes and pathway of RHPS4 to human telomeric G-quadruplex and duplex DNA probed by all-atom molecular dynamics simulations with explicit solvent.

PubMed

Mulholland, Kelly; Siddiquei, Farzana; Wu, Chun

2017-07-19

RHPS4, a potent binder to human telomeric DNA G-quadruplex, shows high efficacy in tumor cell growth inhibition. However, it's preferential binding to DNA G-quadruplex over DNA duplex (about 10 fold) remains to be improved toward its clinical application. A high resolution structure of the single-stranded telomeric DNA G-quadruplexes, or B-DNA duplex, in complex with RHPS4 is not available yet, and the binding nature of this ligand to these DNA forms remains to be elusive. In this study, we carried out 40 μs molecular dynamics binding simulations with a free ligand to decipher the binding pathway of RHPS4 to a DNA duplex and three G-quadruplex folders (parallel, antiparallel and hybrid) of the human telomeric DNA sequence. The most stable binding mode identified for the duplex, parallel, antiparallel and hybrid G-quadruplexes is an intercalation, bottom stacking, top intercalation and bottom intercalation mode, respectively. The intercalation mode with similar binding strength to both the duplex and the G-quadruplexes, explains the lack of binding selectivity of RHPS4 to the G-quadruplex form. Therefore, a ligand modification that destabilizes the duplex intercalation mode but stabilizes the G-quadruplex intercalation mode will improve the binding selectivity toward G-quadruplex. The intercalation mode of RHPS4 to both the duplex and the antiparallel and the hybrid G-quadruplex follows a base flipping-insertion mechanism rather than an open-insertion mechanism. The groove binding, the side binding and the intercalation with flipping out of base were observed to be intermediate states before the full intercalation state with paired bases.

Interaction of DNA with Simple and Mixed Ligand Copper(II) Complexes of 1,10-Phenanthrolines as Studied by DNA-Fiber EPR Spectroscopy

PubMed Central

Chikira, Makoto; Ng, Chew Hee; Palaniandavar, Mallayan

2015-01-01

The interaction of simple and ternary Cu(II) complexes of 1,10-phenanthrolines with DNA has been studied extensively because of their various interesting and important functions such as DNA cleavage activity, cytotoxicity towards cancer cells, and DNA based asymmetric catalysis. Such functions are closely related to the DNA binding modes of the complexes such as intercalation, groove binding, and electrostatic surface binding. A variety of spectroscopic methods have been used to study the DNA binding mode of the Cu(II) complexes. Of all these methods, DNA-fiber electron paramagnetic resonance (EPR) spectroscopy affords unique information on the DNA binding structures of the complexes. In this review we summarize the results of our DNA-fiber EPR studies on the DNA binding structure of the complexes and discuss them together with the data accumulated by using other measurements. PMID:26402668

Crystal Structure of Heart 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase (PFKFB2) and the Inhibitory Influence of Citrate on Substrate Binding

PubMed Central

Crochet, Robert B.; Kim, Jeong-Do; Lee, Herie; Yim, Young-Sun; Kim, Song-Gun; Neau, David; Lee, Yong-Hwan

2016-01-01

The heart-specific isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8Å, respectively. Citrate, a TCA cycle intermediate and well-known inhibitor of PFKFB2, co-crystallized in the 2-kinase domains of both orthologues, occupying the fructose-6-phosphate binding-site and extending into the γ-phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ-phosphate site by citrate proved highly consequential to the binding of co-complexed ATP analogues. The bovine structure, which co-crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co-complexed with AMPPNP, which, unlike ADP, contains a γ-phosphate. The presence of this γ-phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding-pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate’s inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate’s negative feed-back loop of the glycolytic pathway via PFKFB2. PMID:27802586

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

PubMed

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

2018-02-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

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

Dahms, Sven O., E-mail: sdahms@fli-leibniz.de; Mayer, Magnus C.; Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects variousmore » (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.« less

Estrogen Receptor Folding Modulates cSrc Kinase SH2 Interaction via a Helical Binding Mode.

PubMed

Nieto, Lidia; Tharun, Inga M; Balk, Mark; Wienk, Hans; Boelens, Rolf; Ottmann, Christian; Milroy, Lech-Gustav; Brunsveld, Luc

2015-11-20

The estrogen receptors (ERs) feature, next to their transcriptional role, important nongenomic signaling actions, with emerging clinical relevance. The Src Homology 2 (SH2) domain mediated interaction between cSrc kinase and ER plays a key role in this; however the molecular determinants of this interaction have not been elucidated. Here, we used phosphorylated ER peptide and semisynthetic protein constructs in a combined biochemical and structural study to, for the first time, provide a quantitative and structural characterization of the cSrc SH2-ER interaction. Fluorescence polarization experiments delineated the SH2 binding motif in the ER sequence. Chemical shift perturbation analysis by nuclear magnetic resonance (NMR) together with molecular dynamics (MD) simulations allowed us to put forward a 3D model of the ER-SH2 interaction. The structural basis of this protein-protein interaction has been compared with that of the high affinity SH2 binding sequence GpYEEI. The ER features a different binding mode from that of the "two-pronged plug two-hole socket" model in the so-called specificity determining region. This alternative binding mode is modulated via the folding of ER helix 12, a structural element directly C-terminal of the key phosphorylated tyrosine. The present findings provide novel molecular entries for understanding nongenomic ER signaling and targeting the corresponding disease states.

FGFR1 Kinase Inhibitors: Close Regioisomers Adopt Divergent Binding Modes and Display Distinct Biophysical Signatures.

PubMed

Klein, Tobias; Tucker, Julie; Holdgate, Geoffrey A; Norman, Richard A; Breeze, Alexander L

2014-02-13

The binding of a ligand to its target protein is often accompanied by conformational changes of both the protein and the ligand. This is of particular interest, since structural rearrangements of the macromolecular target and the ligand influence the free energy change upon complex formation. In this study, we use X-ray crystallography, isothermal titration calorimetry, and surface-plasmon resonance biosensor analysis to investigate the binding of pyrazolylaminopyrimidine inhibitors to FGFR1 tyrosine kinase, an important anticancer target. Our results highlight that structurally close analogs of this inhibitor series interact with FGFR1 with different binding modes, which are a consequence of conformational changes in both the protein and the ligand as well as the bound water network. Together with the collected kinetic and thermodynamic data, we use the protein-ligand crystal structure information to rationalize the observed inhibitory potencies on a molecular level.

Deciphering structure-activity relationships in a series of Tat/TAR inhibitors.

PubMed

Pascale, Lise; González, Alejandro López; Di Giorgio, Audrey; Gaysinski, Marc; Teixido Closa, Jordi; Tejedor, Roger Estrada; Azoulay, Stéphane; Patino, Nadia

2016-11-01

A series of pentameric "Polyamide Amino Acids" (PAAs) compounds derived from the same trimeric precursor have been synthesized and investigated as HIV TAR RNA ligands, in the absence and in the presence of a Tat fragment. All PAAs bind TAR with similar sub-micromolar affinities but their ability to compete efficiently with the Tat fragment strongly differs, IC50 ranging from 35 nM to >2 μM. While NMR and CD studies reveal that all PAA interact with TAR at the same site and induce globally the same RNA conformational change upon binding, a comparative thermodynamic study of PAA/TAR equilibria highlights distinct TAR binding modes for Tat competitor and non-competitor PAAs. This led us to suggest two distinct interaction modes that have been further validated by molecular modeling studies. While the binding of Tat competitor PAAs induces a contraction at the TAR bulge region, the binding of non-competitor ones widens it. This could account for the distinct PAA ability to compete with Tat fragment. Our work illustrates how comparative thermodynamic studies of a series of RNA ligands of same chemical family are of value for understanding their binding modes and for rationalizing structure-activity relationships.

Structural basis for binding of fluorinated glucose and galactose to Trametes multicolor pyranose 2-oxidase variants with improved galactose conversion.

PubMed

Tan, Tien Chye; Spadiut, Oliver; Gandini, Rosaria; Haltrich, Dietmar; Divne, Christina

2014-01-01

Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the α-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the β-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the β-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose α-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts.

Structural Basis for Binding of Fluorinated Glucose and Galactose to Trametes multicolor Pyranose 2-Oxidase Variants with Improved Galactose Conversion

PubMed Central

Gandini, Rosaria; Haltrich, Dietmar; Divne, Christina

2014-01-01

Each year, about six million tons of lactose are generated from liquid whey as industrial byproduct, and optimally this large carbohydrate waste should be used for the production of value-added products. Trametes multicolor pyranose 2-oxidase (TmP2O) catalyzes the oxidation of various monosaccharides to the corresponding 2-keto sugars. Thus, a potential use of TmP2O is to convert the products from lactose hydrolysis, D-glucose and D-galactose, to more valuable products such as tagatose. Oxidation of glucose is however strongly favored over galactose, and oxidation of both substrates at more equal rates is desirable. Characterization of TmP2O variants (H450G, V546C, H450G/V546C) with improved D-galactose conversion has been given earlier, of which H450G displayed the best relative conversion between the substrates. To rationalize the changes in conversion rates, we have analyzed high-resolution crystal structures of the aforementioned mutants with bound 2- and 3-fluorinated glucose and galactose. Binding of glucose and galactose in the productive 2-oxidation binding mode is nearly identical in all mutants, suggesting that this binding mode is essentially unaffected by the mutations. For the competing glucose binding mode, enzyme variants carrying the H450G replacement stabilize glucose as the α-anomer in position for 3-oxidation. The backbone relaxation at position 450 allows the substrate-binding loop to fold tightly around the ligand. V546C however stabilize glucose as the β-anomer using an open loop conformation. Improved binding of galactose is enabled by subtle relaxation effects at key active-site backbone positions. The competing binding mode for galactose 2-oxidation by V546C stabilizes the β-anomer for oxidation at C1, whereas H450G variants stabilize the 3-oxidation binding mode of the galactose α-anomer. The present study provides a detailed description of binding modes that rationalize changes in the relative conversion rates of D-glucose and D-galactose and can be used to refine future enzyme designs for more efficient use of lactose-hydrolysis byproducts. PMID:24466218

Binding Mode and Structure-Activity Relationships of ITE as an Aryl Hydrocarbon Receptor (AhR) Agonist.

PubMed

Dolciami, Daniela; Gargaro, Marco; Cerra, Bruno; Scalisi, Giulia; Bagnoli, Luana; Servillo, Giuseppe; Fazia, Maria Agnese Della; Puccetti, Paolo; Quintana, Francisco J; Fallarino, Francesca; Macchiarulo, Antonio

2018-02-06

Discovered as a modulator of the toxic response to environmental pollutants, aryl hydrocarbon receptor (AhR) has recently gained attention for its involvement in various physiological and pathological pathways. AhR is a ligand-dependent transcription factor activated by a large array of chemical compounds, which include metabolites of l-tryptophan (l-Trp) catabolism as endogenous ligands of the receptor. Among these, 2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylic acid methyl ester (ITE) has attracted interest in the scientific community, being endowed with nontoxic, immunomodulatory, and anticancer AhR-mediated functions. So far, no information about the binding mode and interactions of ITE with AhR is available. In this study, we used docking and molecular dynamics to propose a putative binding mode of ITE into the ligand binding pocket of AhR. Mutagenesis studies were then instrumental in validating the proposed binding mode, identifying His 285 and Tyr 316 as important key residues for ligand-dependent receptor activation. Finally, a set of ITE analogues was synthesized and tested to further probe molecular interactions of ITE to AhR and characterize the relevance of specific functional groups in the chemical structure for receptor activity. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

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

2012-01-01

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

A combination of spin diffusion methods for the determination of protein-ligand complex structural ensembles.

PubMed

Pilger, Jens; Mazur, Adam; Monecke, Peter; Schreuder, Herman; Elshorst, Bettina; Bartoschek, Stefan; Langer, Thomas; Schiffer, Alexander; Krimm, Isabelle; Wegstroth, Melanie; Lee, Donghan; Hessler, Gerhard; Wendt, K-Ulrich; Becker, Stefan; Griesinger, Christian

2015-05-26

Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinase A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists.

EPA Science Inventory

The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

Structure-Based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists

EPA Science Inventory

The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes.

PubMed

Ni, Lisheng; Jensen, Slade O; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M; Guan, Fiona H X; Brown, Melissa H; Skurray, Ronald A; Firth, Neville; Schumacher, Maria A

2009-11-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon-helix-helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes

PubMed Central

Ni, Lisheng; Jensen, Slade O.; Ky Tonthat, Nam; Berg, Tracey; Kwong, Stephen M.; Guan, Fiona H. X.; Brown, Melissa H.; Skurray, Ronald A.; Firth, Neville; Schumacher, Maria A.

2009-01-01

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon–helix–helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes. PMID:19759211

Effect of Zn2+ binding and enzyme active site on the transition state for RNA 2′-O-transphosphorylation interpreted through kinetic isotope effects

PubMed Central

Chen, Haoyuan; Piccirilli, Joseph A.; Harris, Michael E.; York, Darrin M.

2016-01-01

Divalent metal ions, due to their ability to stabilize high concentrations of negative charge, are important for RNA folding and catalysis. Detailed models derived from the structures and kinetics of enzymes and from computational simulations have been developed. However, in most cases the specific catalytic modes involving metal ions and their mechanistic roles and effects on transition state structures remains controversial. Valuable information about the nature of the transition state is provided by measurement of kinetic isotope effects (KIEs). However, KIEs reflect changes in all bond vibrational modes that differ between the ground state and transition state. QM calculations are therefore essential for developing structural models of the transition state and evaluating mechanistic alternatives. Herein, we present computational models for Zn2+ binding to RNA 2′O-transphosphorylation reaction models that aid in the interpretation of KIE experiments. Different Zn2+ binding modes produce distinct KIE signatures, and one binding mode involving two zinc ions is in close agreement with KIEs measured for non-enzymatic catalysis by Zn2+ aquo ions alone. Interestingly, the KIE signatures in this specific model are also very close to those in RNase A catalysis. These results allow a quantitative connection to be made between experimental KIE measurements and transition state structure and bonding, and provide insight into RNA 2′O-transphosphorylation reactions catalyzed by metal ions and enzymes. PMID:25812974

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

Stachel, Shawn J.; Sanders, John M.; Henze, Darrell A.

We have identified several series of small molecule inhibitors of TrkA with unique binding modes. The starting leads were chosen to maximize the structural and binding mode diversity derived from a high throughput screen of our internal compound collection. These leads were optimized for potency and selectivity employing a structure based drug design approach adhering to the principles of ligand efficiency to maximize binding affinity without overly relying on lipophilic interactions. This endeavor resulted in the identification of several small molecule pan-Trk inhibitor series that exhibit high selectivity for TrkA/B/C versus a diverse panel of kinases. We have also demonstratedmore » efficacy in both inflammatory and neuropathic pain models upon oral dosing. Herein we describe the identification process, hit-to-lead progression, and binding profiles of these selective pan-Trk kinase inhibitors.« less

Structural characterization of nonactive site, TrkA-selective kinase inhibitors

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

Su, Hua-Poo; Rickert, Keith; Burlein, Christine

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residuesmore » from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of—but adjacent to—the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.« less

Structural basis for the antifolding activity of a molecular chaperone

NASA Astrophysics Data System (ADS)

Huang, Chengdong; Rossi, Paolo; Saio, Tomohide; Kalodimos, Charalampos G.

2016-09-01

Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.

Performance of MDockPP in CAPRI rounds 28-29 and 31-35 including the prediction of water-mediated interactions.

PubMed

Xu, Xianjin; Qiu, Liming; Yan, Chengfei; Ma, Zhiwei; Grinter, Sam Z; Zou, Xiaoqin

2017-03-01

Protein-protein interactions are either through direct contacts between two binding partners or mediated by structural waters. Both direct contacts and water-mediated interactions are crucial to the formation of a protein-protein complex. During the recent CAPRI rounds, a novel parallel searching strategy for predicting water-mediated interactions is introduced into our protein-protein docking method, MDockPP. Briefly, a FFT-based docking algorithm is employed in generating putative binding modes, and an iteratively derived statistical potential-based scoring function, ITScorePP, in conjunction with biological information is used to assess and rank the binding modes. Up to 10 binding modes are selected as the initial protein-protein complex structures for MD simulations in explicit solvent. Water molecules near the interface are clustered based on the snapshots extracted from independent equilibrated trajectories. Then, protein-ligand docking is employed for a parallel search for water molecules near the protein-protein interface. The water molecules generated by ligand docking and the clustered water molecules generated by MD simulations are merged, referred to as the predicted structural water molecules. Here, we report the performance of this protocol for CAPRI rounds 28-29 and 31-35 containing 20 valid docking targets and 11 scoring targets. In the docking experiments, we predicted correct binding modes for nine targets, including one high-accuracy, two medium-accuracy, and six acceptable predictions. Regarding the two targets for the prediction of water-mediated interactions, we achieved models ranked as "excellent" in accordance with the CAPRI evaluation criteria; one of these two targets is considered as a difficult target for structural water prediction. Proteins 2017; 85:424-434. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

sc-PDB: a 3D-database of ligandable binding sites—10 years on

PubMed Central

Desaphy, Jérémy; Bret, Guillaume; Rognan, Didier; Kellenberger, Esther

2015-01-01

The sc-PDB database (available at http://bioinfo-pharma.u-strasbg.fr/scPDB/) is a comprehensive and up-to-date selection of ligandable binding sites of the Protein Data Bank. Sites are defined from complexes between a protein and a pharmacological ligand. The database provides the all-atom description of the protein, its ligand, their binding site and their binding mode. Currently, the sc-PDB archive registers 9283 binding sites from 3678 unique proteins and 5608 unique ligands. The sc-PDB database was publicly launched in 2004 with the aim of providing structure files suitable for computational approaches to drug design, such as docking. During the last 10 years we have improved and standardized the processes for (i) identifying binding sites, (ii) correcting structures, (iii) annotating protein function and ligand properties and (iv) characterizing their binding mode. This paper presents the latest enhancements in the database, specifically pertaining to the representation of molecular interaction and to the similarity between ligand/protein binding patterns. The new website puts emphasis in pictorial analysis of data. PMID:25300483

Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes.

PubMed

Jeeva, Subbiah; Pador, Sean; Voss, Brittany; Ganaie, Safder Saieed; Mir, Mohammad Ayoub

2017-01-01

Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5' and 3' termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.

The Binding Mode of the Sonic Hedgehog Inhibitor Robotnikinin, a combined Docking and QM/MM MD Study.

NASA Astrophysics Data System (ADS)

Hitzenberger, Manuel; Schuster, Daniela; Hofer, Thomas S.

2017-10-01

Erroneous activation of the Hedgehog pathway has been linked to a great amount of cancerous diseases and therefore a large number of studies aiming at its inhibition have been carried out. One leverage point for novel therapeutic strategies targeting the proteins involved, is the prevention of complex formation between the extracellular signaling protein Sonic Hedgehog and the transmembrane protein Patched 1. In 2009 robotnikinin, a small molecule capable of binding to and inhibiting the activity of Sonic Hedgehog has been identified, however in the absence of X-ray structures of the Sonic Hedgehog-robotnikinin complex, the binding mode of this inhibitor remains unknown. In order to aid with the identification of novel Sonic Hedgehog inhibitors, the presented investigation elucidates the binding mode of robotnikinin by performing an extensive docking study, including subsequent molecular mechanical as well as quantum mechanical/molecular mechanical molecular dynamics simulations. The attained configurations enabled the identification of a number of key protein-ligand interactions, aiding complex formation and providing stabilizing contributions to the binding of the ligand. The predicted structure of the Sonic Hedgehog-robotnikinin complex is provided via a PDB file as supplementary material and can be used for further reference.

Pyrrole-Based Antitubulin Agents: Two Distinct Binding Modalities Are Predicted for C-2 Analogues in the Colchicine Site

PubMed Central

2011-01-01

3,5-Dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester is a promising antitubulin lead agent that targets the colchicine site of tubulin. C-2 analogues were synthesized and tested for microtubule depolymerizing and antiproliferative activity. Molecular modeling studies using both GOLD docking and HINT (Hydropathic INTeraction) scoring revealed two distinct binding modes that explain the structure–activity relationships and are in accord with the structural basis of colchicine binding to tubulin. The binding mode of higher activity compounds is buried deeper in the site and overlaps well with rings A and C of colchicine, while the lower activity binding mode shows fewer critical contacts with tubulin. The model distinguishes highly active compounds from those with weaker activities and provides novel insights into the colchicine site and compound design. PMID:22611477

Inhibitor-binding mode of homobelactosin C to proteasomes: New insights into class I MHC ligand generation

PubMed Central

Groll, Michael; Larionov, Oleg V.; Huber, Robert; de Meijere, Armin

2006-01-01

Most class I MHC ligands are generated from the vast majority of cellular proteins by proteolysis within the ubiquitin–proteasome pathway and are presented on the cell surface by MHC class I molecules. Here, we present the crystallographic analysis of yeast 20S proteasome in complex with the inhibitor homobelactosin C. The structure reveals a unique inhibitor-binding mode and provides information about the composition of proteasomal primed substrate-binding sites. IFN-γ inducible substitution of proteasomal constitutive subunits by immunosubunits modulates characteristics of generated peptides, thus producing fragments with higher preference for binding to MHC class I molecules. The structural data for the proteasome:homobelactosin C complex provide an explanation for involvement of immunosubunits in antigen generation and open perspectives for rational design of ligands, inhibiting exclusively constitutive proteasomes or immunoproteasomes. PMID:16537370

Effect of Zn2+ binding and enzyme active site on the transition state for RNA 2'-O-transphosphorylation interpreted through kinetic isotope effects.

PubMed

Chen, Haoyuan; Piccirilli, Joseph A; Harris, Michael E; York, Darrin M

2015-11-01

Divalent metal ions, due to their ability to stabilize high concentrations of negative charge, are important for RNA folding and catalysis. Detailed models derived from the structures and kinetics of enzymes and from computational simulations have been developed. However, in most cases the specific catalytic modes involving metal ions and their mechanistic roles and effects on transition state structures remain controversial. Valuable information about the nature of the transition state is provided by measurement of kinetic isotope effects (KIEs). However, KIEs reflect changes in all bond vibrational modes that differ between the ground state and transition state. QM calculations are therefore essential for developing structural models of the transition state and evaluating mechanistic alternatives. Herein, we present computational models for Zn2+ binding to RNA 2'O-transphosphorylation reaction models that aid in the interpretation of KIE experiments. Different Zn2+ binding modes produce distinct KIE signatures, and one binding mode involving two zinc ions is in close agreement with KIEs measured for non-enzymatic catalysis by Zn2+ aquo ions alone. Interestingly, the KIE signatures in this specific model are also very close to those in RNase A catalysis. These results allow a quantitative connection to be made between experimental KIE measurements and transition state structure and bonding, and provide insight into RNA 2'O-ransphosphorylation reactions catalyzed by metal ions and enzymes. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment. Copyright © 2015. Published by Elsevier B.V.

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

PubMed

Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Flexible docking of a ligand peptide to a receptor protein by multicanonical molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Nakajima, Nobuyuki; Higo, Junichi; Kidera, Akinori; Nakamura, Haruki

1997-10-01

A new method for flexible docking by multicanonical molecular dynamics simulation is presented. The method was applied to the binding of a short proline-rich peptide to a Src homology 3 (SH3) domain. The peptide and the side-chains at the ligand binding cleft of SH3 were completely flexible and the large number of possible conformations and dispositions of the peptide were sampled. The reweighted canonical resemble at 300 K resulted in only a few predominant binding modes, one of which was similar to the complex crystal structure. The inverted peptide orientation was also observed in the other binding modes.

Vibrational Softening of a Protein on Ligand Binding

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

Balog, Erica; Perahia, David; Smith, Jeremy C

2011-01-01

Neutron scattering experiments have demonstrated that binding of the cancer drug methotrexate softens the low-frequency vibrations of its target protein, dihydrofolate reductase (DHFR). Here, this softening is fully reproduced using atomic detail normal-mode analysis. Decomposition of the vibrational density of states demonstrates that the largest contributions arise from structural elements of DHFR critical to stability and function. Mode-projection analysis reveals an increase of the breathing-like character of the affected vibrational modes consistent with the experimentally observed increased adiabatic compressibility of the protein on complexation.

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

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

Stanek, Kimberly A.; Patterson-West, Jennifer; Randolph, Peter S.

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homologmore » has been identified in the phylogenetically deep-branching thermophileAquifex aeolicus(Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore,AaeHfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures ofAaeHfq were determined in space groupsP1 andP6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U 6RNA reveals that the outer rim of theAaeHfq hexamer features a well defined binding pocket that is selective for uracil. ThisAaeHfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.« less

Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

NASA Astrophysics Data System (ADS)

Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

2013-11-01

The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

sc-PDB: a 3D-database of ligandable binding sites--10 years on.

PubMed

Desaphy, Jérémy; Bret, Guillaume; Rognan, Didier; Kellenberger, Esther

2015-01-01

The sc-PDB database (available at http://bioinfo-pharma.u-strasbg.fr/scPDB/) is a comprehensive and up-to-date selection of ligandable binding sites of the Protein Data Bank. Sites are defined from complexes between a protein and a pharmacological ligand. The database provides the all-atom description of the protein, its ligand, their binding site and their binding mode. Currently, the sc-PDB archive registers 9283 binding sites from 3678 unique proteins and 5608 unique ligands. The sc-PDB database was publicly launched in 2004 with the aim of providing structure files suitable for computational approaches to drug design, such as docking. During the last 10 years we have improved and standardized the processes for (i) identifying binding sites, (ii) correcting structures, (iii) annotating protein function and ligand properties and (iv) characterizing their binding mode. This paper presents the latest enhancements in the database, specifically pertaining to the representation of molecular interaction and to the similarity between ligand/protein binding patterns. The new website puts emphasis in pictorial analysis of data. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of Myo7b/USH1C complex suggests a general PDZ domain binding mode by MyTH4-FERM myosins

PubMed Central

Li, Jianchao; He, Yunyun; Weck, Meredith L.; Lu, Qing; Tyska, Matthew J.; Zhang, Mingjie

2017-01-01

Unconventional myosin 7a (Myo7a), myosin 7b (Myo7b), and myosin 15a (Myo15a) all contain MyTH4-FERM domains (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in their cargo binding tails and are essential for the growth and function of microvilli and stereocilia. Numerous mutations have been identified in the MyTH4-FERM tandems of these myosins in patients suffering visual and hearing impairment. Although a number of MF domain binding partners have been identified, the molecular basis of interactions with the C-terminal MF domain (CMF) of these myosins remains poorly understood. Here we report the high-resolution crystal structure of Myo7b CMF in complex with the extended PDZ3 domain of USH1C (a.k.a., Harmonin), revealing a previously uncharacterized interaction mode both for MyTH4-FERM tandems and for PDZ domains. We predicted, based on the structure of the Myo7b CMF/USH1C PDZ3 complex, and verified that Myo7a CMF also binds to USH1C PDZ3 using a similar mode. The structure of the Myo7b CMF/USH1C PDZ complex provides mechanistic explanations for >20 deafness-causing mutations in Myo7a CMF. Taken together, these findings suggest that binding to PDZ domains, such as those from USH1C, PDZD7, and Whirlin, is a common property of CMFs of Myo7a, Myo7b, and Myo15a. PMID:28439001

Structural insight into TPX2-stimulated microtubule assembly

PubMed Central

2017-01-01

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements (’ridge’ and ‘wedge’) in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation. PMID:29120325

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

PubMed Central

Clifton, Matthew C.; Dranow, David M.; Leed, Alison; Fulroth, Ben; Fairman, James W.; Abendroth, Jan; Atkins, Kateri A.; Wallace, Ellen; Fan, Dazhong; Xu, Guoping; Ni, Z. J.; Daniels, Doug; Van Drie, John; Wei, Guo; Burgin, Alex B.; Golub, Todd R.; Hubbard, Brian K.; Serrano-Wu, Michael H.

2015-01-01

Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors. PMID:25909780

Molecular modeling studies of substrate binding by penicillin acylase.

PubMed

Chilov, G G; Stroganov, O V; Svedas, V K

2008-01-01

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.

Cooperative binding modes of Cu(II) in prion protein

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

2007-03-01

The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

Binding Modes of Thioflavin T Molecules to Prion Peptide Assemblies Identified by Using Scanning Tunneling Microscopy

PubMed Central

2011-01-01

The widely used method to monitor the aggregation process of amyloid peptide is thioflavin T (ThT) assay, while the detailed molecular mechanism is still not clear. In this work, we report here the direct identification of the binding modes of ThT molecules with the prion peptide GNNQQNY by using scanning tunneling microscopy (STM). The assembly structures of GNNQQNY were first observed by STM on a graphite surface, and the introduction of ThT molecules to the surface facilitated the STM observations of the adsorption conformations of ThT with peptide strands. ThT molecules are apt to adsorb on the peptide assembly with β-sheet structure and oriented parallel with the peptide strands adopting four different binding modes. This effort could benefit the understanding of the mechanisms of the interactions between labeling species or inhibitory ligands and amyloid peptides, which is keenly needed for developing diagnostic and therapeutic approaches. PMID:22778872

Structural Biology Guides Antibiotic Discovery

ERIC Educational Resources Information Center

Polyak, Steven

2014-01-01

Modern drug discovery programs require the contribution of researchers in a number of specialist areas. One of these areas is structural biology. Using X-ray crystallography, the molecular basis of how a drug binds to its biological target and exerts its mode of action can be defined. For example, a drug that binds into the active site of an…

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

PubMed

Ha, Sung Chul; Choi, Jongkeun; Hwang, Hye-Yeon; Rich, Alexander; Kim, Yang-Gyun; Kim, Kyeong Kyu

2009-02-01

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.

Atomic elucidation of the cyclodextrin effects on DDT solubility and biodegradation.

PubMed

Ren, Baiping; Zhang, Mingzhen; Gao, Huipeng; Zheng, Jie; Jia, Lingyun

2016-07-14

DDT (1,1,1-trichloro-2.2-bis(p-chlorophenyl)ethane), one of the most abused insecticides, is a highly hazardous component for both human health and environmental applications. The biodegradation of DDT into non-toxic, environmentally benign components is strongly limited by the poor bioavailability of DDT. In this work, we combined experiments and molecular simulations to examine the effect of three cyclodextrins (α-, β-, and γ-CD) on their structure-specific interactions with DDT, specifically in relation to DDT solubility and biodegradability. It was found that all three CDs were able to bind to DDT with their inner hydrophobic cavity and different binding affinities and orientations, demonstrating their ability to improve DDT solubility. Different from the strong binding between DDT and β-/γ-CDs via a fully DDT bury mode, α-CD had a relatively weak binding with DDT via a partial DDT bury mode, which allowed DDT to be readily disassociated from α-CD at the lipid membrane interface, followed by DDT permeation into and across the cell membrane. The different binding modes between DDT and CDs explain why only α-CD can promote the bioavailability and biodegradation of DDT by simultaneously increasing its aqueous solubility and membrane interaction. This work provides structural-based binding information for the further modification and optimization of these three CDs to enhance their solubility and biodegradability of DDT.

Dynamics of the Extended String-Like Interaction of TFIIE with the p62 Subunit of TFIIH.

PubMed

Okuda, Masahiko; Higo, Junichi; Komatsu, Tadashi; Konuma, Tsuyoshi; Sugase, Kenji; Nishimura, Yoshifumi

2016-09-06

General transcription factor II E (TFIIE) contains an acid-rich region (residues 378-393) in its α-subunit, comprising 13 acidic and two hydrophobic (Phe387 and Val390) residues. Upon binding to the p62 subunit of TFIIH, the acidic region adopts an extended string-like structure on the basic groove of the pleckstrin homology domain (PHD) of p62, and inserts Phe387 and Val390 into two shallow pockets in the groove. Here, we have examined the dynamics of this interaction by NMR and molecular dynamics (MD) simulations. Although alanine substitution of Phe387 and/or Val390 greatly reduced binding to PHD, the binding mode of the mutants was similar to that of the wild-type, as judged by the chemical-shift changes of the PHD. NMR relaxation dispersion profiles of the interaction exhibited large amplitudes for residues in the C-terminal half-string in the acidic region (Phe387, Glu388, Val390, Ala391, and Asp392), indicating a two-site binding mode: one corresponding to the final complex structure, and one to an off-pathway minor complex. To probe the off-pathway complex structure, an atomically detailed free-energy landscape of the binding mode was computed by all-atom multicanonical MD. The most thermodynamically stable cluster corresponded to the final complex structure. One of the next stable clusters was the off-pathway structure cluster, showing the reversed orientation of the C-terminal half-string on the PHD groove, as compared with the final structure. MD calculations elucidated that the C-terminal half-acidic-string forms encounter complexes mainly around the positive groove region with nearly two different orientations of the string, parallel and antiparallel to the final structure. Interestingly, the most encountered complexes exhibit a parallel-like orientation, suggesting that the string has a tendency to bind around the groove in the proper orientation with the aid of Phe387 and/or Val390 to proceed smoothly to the final complex structure. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Crystal Structure of the Arginine Repressor Protein in Complex With the DNA Operator From Mycobacterium Tuberculosis

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

Cherney, L.T.; Cherney, M.M.; Garen, C.R.

2009-05-12

The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the L-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of arginine repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 {angstrom} resolution with bound arginine and at 2.15 {angstrom} resolution in the unliganded form. These structures show that six molecules ofmore » MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11{sup o} upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.« less

Exploration of multiple Sortase A protein conformations in virtual screening

NASA Astrophysics Data System (ADS)

Gao, Chunxia; Uzelac, Ivana; Gottfries, Johan; Eriksson, Leif A.

2016-02-01

Methicillin resistant Staphylococcus aureus (MRSA) has become a major health concern which has brought about an urgent need for new therapeutic agents. As the S. aureus Sortase A (SrtA) enzyme contributes to the adherence of the bacteria to the host cells, inhibition thereof by small molecules could be employed as potential antivirulence agents, also towards resistant strains. Albeit several virtual docking SrtA campaigns have been reported, no strongly inhibitatory non-covalent binders have as yet emerged therefrom. In order to better understand the binding modes of small molecules, and the effect of different receptor structures employed in the screening, we herein report on an exploratory study employing 10 known binders and 500 decoys on 100 SrtA structures generated from regular or steered molecular dynamics simulations on four different SrtA crystal/NMR structures. The results suggest a correlation between the protein structural flexibility and the virtual screening performance, and confirm the noted immobilization of the β6/β7 loop upon substrate binding. The NMR structures reported appear to perform slightly better than the Xray-crystal structures, but the binding modes fluctuate tremendously, and it might be suspected that the catalytic site is not necessarily the preferred site of binding for some of the reported active compounds.

Exploration of multiple Sortase A protein conformations in virtual screening

PubMed Central

Gao, Chunxia; Uzelac, Ivana; Gottfries, Johan; Eriksson, Leif A.

2016-01-01

Methicillin resistant Staphylococcus aureus (MRSA) has become a major health concern which has brought about an urgent need for new therapeutic agents. As the S. aureus Sortase A (SrtA) enzyme contributes to the adherence of the bacteria to the host cells, inhibition thereof by small molecules could be employed as potential antivirulence agents, also towards resistant strains. Albeit several virtual docking SrtA campaigns have been reported, no strongly inhibitatory non-covalent binders have as yet emerged therefrom. In order to better understand the binding modes of small molecules, and the effect of different receptor structures employed in the screening, we herein report on an exploratory study employing 10 known binders and 500 decoys on 100 SrtA structures generated from regular or steered molecular dynamics simulations on four different SrtA crystal/NMR structures. The results suggest a correlation between the protein structural flexibility and the virtual screening performance, and confirm the noted immobilization of the β6/β7 loop upon substrate binding. The NMR structures reported appear to perform slightly better than the Xray-crystal structures, but the binding modes fluctuate tremendously, and it might be suspected that the catalytic site is not necessarily the preferred site of binding for some of the reported active compounds. PMID:26846342

Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent

PubMed Central

Bonenfant, Débora; Rubert, Joëlle; Vangrevelinghe, Eric; Scheufler, Clemens; Marque, Fanny; Régnier, Catherine H.; De Pover, Alain; Ryckelynck, Hugues; Bhagwat, Neha; Koppikar, Priya; Goel, Aviva; Wyder, Lorenza; Tavares, Gisele; Baffert, Fabienne; Pissot-Soldermann, Carole; Manley, Paul W.; Gaul, Christoph; Voshol, Hans; Levine, Ross L.; Sellers, William R.; Hofmann, Francesco; Radimerski, Thomas

2016-01-01

JAK inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type-I binding mode leads to an increase in JAK activation-loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type-II inhibition acts in the opposite manner, leading to a loss of activation-loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation-loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation-loop may or may not be elicited. PMID:22684457

Anthranilate phosphoribosyltransferase: Binding determinants for 5'-phospho-alpha-d-ribosyl-1'-pyrophosphate (PRPP) and the implications for inhibitor design.

PubMed

Evans, Genevieve L; Furkert, Daniel P; Abermil, Nacim; Kundu, Preeti; de Lange, Katrina M; Parker, Emily J; Brimble, Margaret A; Baker, Edward N; Lott, J Shaun

2018-02-01

Phosphoribosyltransferases (PRTs) bind 5'-phospho-α-d-ribosyl-1'-pyrophosphate (PRPP) and transfer its phosphoribosyl group (PRib) to specific nucleophiles. Anthranilate PRT (AnPRT) is a promiscuous PRT that can phosphoribosylate both anthranilate and alternative substrates, and is the only example of a type III PRT. Comparison of the PRPP binding mode in type I, II and III PRTs indicates that AnPRT does not bind PRPP, or nearby metals, in the same conformation as other PRTs. A structure with a stereoisomer of PRPP bound to AnPRT from Mycobacterium tuberculosis (Mtb) suggests a catalytic or post-catalytic state that links PRib movement to metal movement. Crystal structures of Mtb-AnPRT in complex with PRPP and with varying occupancies of the two metal binding sites, complemented by activity assay data, indicate that this type III PRT binds a single metal-coordinated species of PRPP, while an adjacent second metal site can be occupied due to a separate binding event. A series of compounds were synthesized that included a phosphonate group to probe PRPP binding site. Compounds containing a "bianthranilate"-like moiety are inhibitors with IC 50 values of 10-60μM, and K i values of 1.3-15μM. Structures of Mtb-AnPRT in complex with these compounds indicate that their phosphonate moieties are unable to mimic the binding modes of the PRib or pyrophosphate moieties of PRPP. The AnPRT structures presented herein indicated that PRPP binds a surface cleft and becomes enclosed due to re-positioning of two mobile loops. Copyright © 2017 Elsevier B.V. All rights reserved.

RVX-297- a novel BD2 selective inhibitor of BET bromodomains

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

Kharenko, Olesya A., E-mail: olesya@zenithepigenetics.com; Gesner, Emily M.; Patel, Reena G.

Bromodomains are epigenetic readers that specifically bind to the acetyl lysine residues of histones and transcription factors. Small molecule BET bromodomain inhibitors can disrupt this interaction which leads to potential modulation of several disease states. Here we describe the binding properties of a novel BET inhibitor RVX-297 that is structurally related to the clinical compound RVX-208, currently undergoing phase III clinical trials for the treatment of cardiovascular diseases, but is distinctly different in its biological and pharmacokinetic profiles. We report that RVX-297 preferentially binds to the BD2 domains of the BET bromodomain and Extra Terminal (BET) family of protein. Wemore » demonstrate the differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography, and describe the structural differences driving the BD2 selective binding of RVX-297. The isothermal titration calorimetry (ITC) data illustrate the related differential thermodynamics of binding of RVX-297 to single as well as dual BET bromodomains. - Highlights: • A novel inhibitor of BET bromodomains, RVX-297 is described. • The differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography are described. • RVX-297 preferentially binds to the BD2 domains of the BET bromodomains. • The structural and thermodynamic properties of the BD2 selective binding of RVX-297 are characterized.« less

Dynamic binding of replication protein a is required for DNA repair

PubMed Central

Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

2016-01-01

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

Interaction of thioflavin T with amyloid fibrils: stoichiometry and affinity of dye binding, absorption spectra of bound dye.

PubMed

Sulatskaya, Anna I; Kuznetsova, Irina M; Turoverov, Konstantin K

2011-10-06

The fluorescence of the benzothiazole dye thioflavin T (ThT) is a well-known test for amyloid fibril formation. It has now become evident that ThT can also be used for structural investigations of amyloid fibrils and even for the treatment of amyloid diseases. In this case, one of the most urgent problems is an accurate determination of ThT-amyloid fibril binding parameters: the number of binding modes, stoichiometry, and binding constant for each mode. To obtain information concerning the ThT-amyloid fibril binding parameters, we propose to use absorption spectrophotometry of solutions prepared by equilibrium microdialysis. This approach is inherently designed for the determination of dye-receptor binding parameters. However, it has been very rarely used in the study of dye-protein interactions and has never been used to study the binding parameters of ThT or its analogues to amyloid fibrils. We showed that, when done in corpore, this approach enables the determination of not only binding parameters but also the absorption spectrum and molar extinction coefficient of ThT bound to sites of different binding modes. The proposed approach was used for the examination of lysozyme amyloid fibrils. Two binding modes were found for the ThT-lysozyme amyloid fibril interaction. These binding modes have significantly different binding constants (K(b1) = 7.5 × 10(6) M(-1), K(b2) = 5.6 × 10(4) M(-1)) and a different number of dye binding sites on the amyloid fibrils per protein molecule (n(1) = 0.11, n(2) = 0.24). The absorption spectra of ThT bound to sites of different modes differ from each other (ε(b1,max) = 5.1 × 10(4) M(-1) cm(-1), ε(b2,max) = 6.7 × 10(4) M(-1)cm(-1), λ(max) = 449 nm) and significantly differ from that of free ThT in aqueous solution (ε(max) = 3.2 × 10(4) M(-1)cm(-1), λ(max) = 412 nm). © 2011 American Chemical Society

Binding Modes of Teixobactin to Lipid II: Molecular Dynamics Study.

PubMed

Liu, Yang; Liu, Yaxin; Chan-Park, Mary B; Mu, Yuguang

2017-12-08

Teixobactin (TXB) is a newly discovered antibiotic targeting the bacterial cell wall precursor Lipid II (L II ). In the present work, four binding modes of TXB on L II were identified by a contact-map based clustering method. The highly flexible binary complex ensemble was generated by parallel tempering metadynamics simulation in a well-tempered ensemble (PTMetaD-WTE). In agreement with experimental findings, the pyrophosphate group and the attached first sugar subunit of L II are found to be the minimal motif for stable TXB binding. Three of the four binding modes involve the ring structure of TXB and have relatively higher binding affinities, indicating the importance of the ring motif of TXB in L II recognition. TXB-L II complexes with a ratio of 2:1 are also predicted with configurations such that the ring motif of two TXB molecules bound to the pyrophosphate-MurNAc moiety and the glutamic acid residue of one L II , respectively. Our findings disclose that the ring motif of TXB is critical to L II binding and novel antibiotics can be designed based on its mimetics.

Water-Tryptophan Interactions: Lone-pair⋅⋅⋅π or O-H⋅⋅⋅π? Molecular Dynamics Simulations of β-Galactosidase Suggest that Both Modes Can Co-exist.

PubMed

Durec, Matúš; Marek, Radek; Kozelka, Jiří

2018-04-17

In proteins, the indole side chain of tryptophan can interact with water molecules either in-plane, forming hydrogen bonds, or out-of-plane, with the water molecule contacting the aromatic π face. The latter interaction can be either of the lone pair⋅⋅⋅π (lp⋅⋅⋅π) type or corresponds to the O-H⋅⋅⋅π binding mode, an ambiguity that X-ray structures usually do not resolve. Here, we report molecular dynamics (MD) simulations of a solvated β-galactosidase monomer, which illustrate how a water molecule located at the π face of an indole side chain of tryptophan can adapt to the position of proximate residues and "select" its binding mode. In one such site, the water molecule is predicted to rapidly oscillate between the O-H⋅⋅⋅π and lp⋅⋅⋅π binding modes, thus gaining entropic advantage. Our MD simulations provide support for the role of lp⋅⋅⋅π interactions in the stabilization of protein structures. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations.

PubMed

Ge, Yushu; van der Kamp, Marc; Malaisree, Maturos; Liu, Dan; Liu, Yi; Mulholland, Adrian J

2017-11-01

Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.

Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Ge, Yushu; van der Kamp, Marc; Malaisree, Maturos; Liu, Dan; Liu, Yi; Mulholland, Adrian J.

2017-11-01

Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

PubMed Central

Hanaoka, Shingo; Nagadoi, Aritaka; Nishimura, Yoshifumi

2005-01-01

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2. PMID:15608118

Customizing G Protein-coupled receptor models for structure-based virtual screening.

PubMed

de Graaf, Chris; Rognan, Didier

2009-01-01

This review will focus on the construction, refinement, and validation of G Protein-coupled receptor models for the purpose of structure-based virtual screening. Practical tips and tricks derived from concrete modeling and virtual screening exercises to overcome the problems and pitfalls associated with the different steps of the receptor modeling workflow will be presented. These examples will not only include rhodopsin-like (class A), but also secretine-like (class B), and glutamate-like (class C) receptors. In addition, the review will present a careful comparative analysis of current crystal structures and their implication on homology modeling. The following themes will be discussed: i) the use of experimental anchors in guiding the modeling procedure; ii) amino acid sequence alignments; iii) ligand binding mode accommodation and binding cavity expansion; iv) proline-induced kinks in transmembrane helices; v) binding mode prediction and virtual screening by receptor-ligand interaction fingerprint scoring; vi) extracellular loop modeling; vii) virtual filtering schemes. Finally, an overview of several successful structure-based screening shows that receptor models, despite structural inaccuracies, can be efficiently used to find novel ligands.

Automated docking of ligands to an artificial active site: augmenting crystallographic analysis with computer modeling

NASA Astrophysics Data System (ADS)

Rosenfeld, Robin J.; Goodsell, David S.; Musah, Rabi A.; Morris, Garrett M.; Goodin, David B.; Olson, Arthur J.

2003-08-01

The W191G cavity of cytochrome c peroxidase is useful as a model system for introducing small molecule oxidation in an artificially created cavity. A set of small, cyclic, organic cations was previously shown to bind in the buried, solvent-filled pocket created by the W191G mutation. We docked these ligands and a set of non-binders in the W191G cavity using AutoDock 3.0. For the ligands, we compared docking predictions with experimentally determined binding energies and X-ray crystal structure complexes. For the ligands, predicted binding energies differed from measured values by ± 0.8 kcal/mol. For most ligands, the docking simulation clearly predicted a single binding mode that matched the crystallographic binding mode within 1.0 Å RMSD. For 2 ligands, where the docking procedure yielded an ambiguous result, solutions matching the crystallographic result could be obtained by including an additional crystallographically observed water molecule in the protein model. For the remaining 2 ligands, docking indicated multiple binding modes, consistent with the original electron density, suggesting disordered binding of these ligands. Visual inspection of the atomic affinity grid maps used in docking calculations revealed two patches of high affinity for hydrogen bond donating groups. Multiple solutions are predicted as these two sites compete for polar hydrogens in the ligand during the docking simulation. Ligands could be distinguished, to some extent, from non-binders using a combination of two trends: predicted binding energy and level of clustering. In summary, AutoDock 3.0 appears to be useful in predicting key structural and energetic features of ligand binding in the W191G cavity.

Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

PubMed Central

Peter, Daniel; Weber, Ramona; Köne, Carolin; Chung, Min-Yi; Ebertsch, Linda; Truffault, Vincent; Weichenrieder, Oliver; Igreja, Cátia; Izaurralde, Elisa

2015-01-01

The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E–Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation. PMID:26294658

Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition

PubMed Central

Kumar, Prashant; Reithofer, Viktoria; Reisinger, Manuel; Wallner, Silvia; Pavkov-Keller, Tea; Macheroux, Peter; Gruber, Karl

2016-01-01

Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors. PMID:27025154

Spectroscopic investigation of the interaction between G-quadruplex of KRAS promoter sequence and three isoquinoline alkaloids

NASA Astrophysics Data System (ADS)

Wen, Li-Na; Xie, Meng-Xia

2017-01-01

KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90 ± 0.16) × 106 L mol- 1, (0.93 ± 0.21) × 106 L mol- 1 and (1.16 ± 0.45) × 106 L mol- 1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

Structure of the C-terminal domain of the arginine repressor protein from Mycobacterium tuberculosis

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

Cherney, Leonid T.; Cherney, Maia M.; Garen, Craig R.

2008-09-01

The structure of the core domain of the arginine repressor protein from M. tuberculosis has been determined with (1.85 Å resolution) and without (2.15 Å resolution) the arginine corepressor bound. Three additional arginine molecules have been found to bind to the core domain hexamer at high (0.2 M) arginine concentration. The Mycobacterium tuberculosis (Mtb) gene product encoded by open reading frame Rv1657 is an arginine repressor (ArgR). All genes involved in the l-arginine (hereafter arginine) biosynthetic pathway are essential for optimal growth of the Mtb pathogen, thus making MtbArgR a potential target for drug design. The C-terminal domains of argininemore » repressors (CArgR) participate in oligomerization and arginine binding. Several crystal forms of CArgR from Mtb (MtbCArgR) have been obtained. The X-ray crystal structures of MtbCArgR were determined at 1.85 Å resolution with bound arginine and at 2.15 Å resolution in the unliganded form. These structures show that six molecules of MtbCArgR are arranged into a hexamer having approximate 32 point symmetry that is formed from two trimers. The trimers rotate relative to each other by about 11° upon binding arginine. All residues in MtbCArgR deemed to be important for hexamer formation and for arginine binding have been identified from the experimentally determined structures presented. The hexamer contains six regular sites in which the arginine molecules have one common binding mode and three sites in which the arginine molecules have two overlapping binding modes. The latter sites only bind the ligand at high (200 mM) arginine concentrations.« less

Structural insight into the TFIIE–TFIIH interaction: TFIIE and p53 share the binding region on TFIIH

PubMed Central

Okuda, Masahiko; Tanaka, Aki; Satoh, Manami; Mizuta, Shoko; Takazawa, Manabu; Ohkuma, Yoshiaki; Nishimura, Yoshifumi

2008-01-01

RNA polymerase II and general transcription factors (GTFs) assemble on a promoter to form a transcription preinitiation complex (PIC). Among the GTFs, TFIIE recruits TFIIH to complete the PIC formation and regulates enzymatic activities of TFIIH. However, the mode of binding between TFIIE and TFIIH is poorly understood. Here, we demonstrate the specific binding of the C-terminal acidic domain (AC-D) of the human TFIIEα subunit to the pleckstrin homology domain (PH-D) of the human TFIIH p62 subunit and describe the solution structures of the free and PH-D-bound forms of AC-D. Although the flexible N-terminal acidic tail from AC-D wraps around PH-D, the core domain of AC-D also interacts with PH-D. AC-D employs an entirely novel binding mode, which differs from the amphipathic helix method used by many transcriptional activators. So the binding surface between PH-D and AC-D is much broader than the specific binding surface between PH-D and the p53 acidic fragments. From our in vitro studies, we demonstrate that this interaction could be a switch to replace p53 with TFIIE on TFIIH in transcription. PMID:18354501

Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

PubMed Central

Kaufmann, Kristian W.; Dawson, Eric S.; Henry, L. Keith; Field, Julie R.; Blakely, Randy D.; Meiler, Jens

2009-01-01

To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuTAa) structure reported by Yamashita et al. (Nature 2005;437:215–223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuTAa is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to cHitically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuTAa structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected. PMID:18704946

Proposed Mode of Binding and Action of Positive Allosteric Modulators at Opioid Receptors

PubMed Central

2016-01-01

Available crystal structures of opioid receptors provide a high-resolution picture of ligand binding at the primary (“orthosteric”) site, that is, the site targeted by endogenous ligands. Recently, positive allosteric modulators of opioid receptors have also been discovered, but their modes of binding and action remain unknown. Here, we use a metadynamics-based strategy to efficiently sample the binding process of a recently discovered positive allosteric modulator of the δ-opioid receptor, BMS-986187, in the presence of the orthosteric agonist SNC-80, and with the receptor embedded in an explicit lipid–water environment. The dynamics of BMS-986187 were enhanced by biasing the potential acting on the ligand–receptor distance and ligand–receptor interaction contacts. Representative lowest-energy structures from the reconstructed free-energy landscape revealed two alternative ligand binding poses at an allosteric site delineated by transmembrane (TM) helices TM1, TM2, and TM7, with some participation of TM6. Mutations of amino acid residues at these proposed allosteric sites were found to either affect the binding of BMS-986187 or its ability to modulate the affinity and/or efficacy of SNC-80. Taken together, these combined experimental and computational studies provide the first atomic-level insight into the modulation of opioid receptor binding and signaling by allosteric modulators. PMID:26841170

Deep insights into the mode of ATP-binding mechanism in Zebrafish cyclin-dependent protein kinase-like 1 (zCDKL1): A molecular dynamics approach.

PubMed

Rout, Ajaya Kumar; Dehury, Budheswar; Maharana, Jitendra; Nayak, Chirasmita; Baisvar, Vishwamitra Singh; Behera, Bijay Kumar; Das, Basanta Kumar

2018-05-01

In eukaryotes, the serine/threonine kinases (STKs) belonging to cyclin-dependent protein kinases (CDKs) play significant role in control of cell division and curb transcription in response to several extra and intra-cellular signals indispensable for enzymatic activity. The zebrafish cyclin-dependent protein kinase-like 1 protein (zCDKL1) shares a high degree of sequence and structural similarity with mammalian orthologs and express in brain, ovary, testis, and low levels in other tissues. Regardless of its importance in the developmental process, the structure, function and mode of ATP recognition have not been investigated yet due to lack of experimental data. Henceforth, to gain atomistic insights in to the structural dynamics and mode of ATP binding, a series of computational techniques involving theoretical modeling, docking, molecular dynamics (MD) simulations and MM/PBSA binding free energies were employed. The modeled bi-lobed zCDKL1 shares a high degree of secondary structure topology with human orthologs where ATP prefers to lie in the central cavity of the bi-lobed catalytic domain enclosed by strong hydrogen bonding, electrostatic and hydrophobic contacts. Long range MD simulation portrayed that catalytic domain of zCDKL1 to be highly rigid in nature as compared to the complex (zCDKL1-ATP) form. Comparative analysis with its orthologs revealed that conserved amino acids i.e., Ile10, Gly11, Glu12, Val18, Arg31, Phe80, Glu 130, Cys143 and Asp144 were crucial for ATP binding mechanism, which needs further investigation for legitimacy. MM/PBSA method revealed that van der Waals, electrostatic and polar solvation energy mostly contributes towards negative free energy. The implications of ATP binding mechanism inferred through these structural bioinformatics approaches will help in understanding the catalytic mechanisms of important STKs in eukaryotic system. Copyright © 2018. Published by Elsevier Inc.

Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

PubMed Central

Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y.; Varnado, Brittany; Beutler, John A.; Murelli, Ryan P.; Le Grice, Stuart F. J.; Tang, Liang

2015-01-01

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

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.

Deciphering the mechanism of interaction of edifenphos with calf thymus DNA

NASA Astrophysics Data System (ADS)

Ahmad, Ajaz; Ahmad, Masood

2018-01-01

Edifenphos is an important organophosphate pesticide with many antifungal and anti-insecticidal properties but it may cause potential hazards to human health. In this work, we have tried to explore the binding mode of action and mechanism of edifenphos to calf thymus DNA (CT-DNA). Several experiments such as ultraviolet-visible absorption spectra and emission spectroscopy showed complex formation between edifenphos and CT-DNA and low binding constant values supporting groove binding mode. These results were further confirmed by circular dichroism (CD), CT-DNA melting studies, viscosity measurements, density functional theory and molecular docking. CD study suggests that edifenphos does not alter native structure of CT-DNA. Isothermal calorimetry reveals that binding of edifenphos with CT-DNA is enthalpy driven process. Competitive binding assay and effect of ionic strength showed that edifenphos binds to CT-DNA via groove binding manner. Hence, edifenphos is a minor groove binder preferably interacting with A-T regions with docking score - 6.84 kJ/mol.

Crystal structure analysis of human serum albumin complexed with sodium 4-phenylbutyrate.

PubMed

Kawai, Akito; Yamasaki, Keishi; Enokida, Taisuke; Miyamoto, Shuichi; Otagiri, Masaki

2018-03-01

Sodium 4-phenylbutyrate (PB) is an orphan drug for the treatment of urea cycle disorders. It also inhibits the development of endoplasmic reticulum stress, the action of histone deacetylases and as a regulator of the hepatocanalicular transporter. PB is generally considered to have the potential for use in the treatment of the diseases such as cancer, neurodegenerative diseases and metabolic diseases. In a previous study, we reported that PB is primarily bound to human serum albumin (HSA) in plasma and its binding site is drug site 2. However, details of the binding mode of PB to HSA remain unknown. To address this issue, we examined the crystal structure of HSA with PB bound to it. The structure of the HSA-PB complex indicates that the binding mode of PB to HSA is quite similar to that for octanoate or drugs that bind to drug site 2, as opposed to that for other medium-chain length of fatty acids. These findings provide useful basic information related to drug-HSA interactions. Moreover, the information presented herein is valuable in terms of providing safe and efficient treatment and diagnosis in clinical settings.

Magnetic field effects on charge structure factors of gapped graphene structure

NASA Astrophysics Data System (ADS)

Rezania, Hamed; Tawoose, Nasrin

2018-02-01

We present the behaviors of dynamical and static charge susceptibilities of undoped gapped graphene using the Green's function approach in the context of tight binding model Hamiltonian. Specially, the effects of magnetic field on the plasmon modes of gapped graphene structure are investigated via calculating correlation function of charge density operators. Our results show the increase of magnetic field leads to disappear high frequency plasmon mode for gapped case. We also show that low frequency plasmon mode has not affected by increase of magnetic field and chemical potential. Finally the temperature dependence of static charge structure factor of gapp graphene structure is studied. The effects of both magnetic field and gap parameter on the static structure factor are discusses in details.

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor

PubMed Central

Suzuki, Kano; Mizutani, Kenji; Maruyama, Shintaro; Shimono, Kazumi; Imai, Fabiana L.; Muneyuki, Eiro; Kakinuma, Yoshimi; Ishizuka-Katsura, Yoshiko; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamato, Ichiro; Murata, Takeshi

2016-01-01

V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model. PMID:27807367

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

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

Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya

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

Linear and circular dichroism characterization of thionine binding mode with DNA polynucleotides

NASA Astrophysics Data System (ADS)

Tuite, Eimer Mary; Nordén, Bengt

2018-01-01

The binding mode of thionine (3,7-diamino-5-phenothiazinium) with alternating and non-alternating DNA polynucleotides at low binding ratios was conclusively determined using linear and circular dichroism spectroscopies. The binding to [poly(dG-dC)]2 and poly(dG)·poly(dC) was purely intercalative and was insensitive to ionic strength. Intercalative binding to [poly(dA-dT)]2 is observed at low ionic strength, but a shift of some dye to an non-intercalative mode is observed as the background salt concentration increases. With poly(dA)·poly(dT), intercalative binding is unfavourable, although some dye molecules may intercalate at low ionic strength, and groove binding is strongly promoted with increasing concentration of background salt. However, stacking with bases is observed with single-stranded poly(dA) and with triplex poly(dT)*poly(dA)·poly(dT) which suggests that the unusual structure of poly(dA)·poly(dT) precludes intercalation. Thionine behaves similarly to the related dye methylene blue, and small differences may be attributed either to the ability of thionine to form H-bonds that stabilize intercalation or to its improved stacking interactions in the basepair pocket on steric grounds.

Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes

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

C Simmons; C Magee; D Smith

The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADPmore » cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.« less

A Novel Cofactor-binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity*

PubMed Central

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R.; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-01-01

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5′-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD+ and XMP/NAD+. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD+-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD+-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. PMID:25572472

Theory and Normal Mode Analysis of Change in Protein Vibrational Dynamics on Ligand Binding

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

Mortisugu, Kei; Njunda, Brigitte; Smith, Jeremy C

2009-12-01

The change of protein vibrations on ligand binding is of functional and thermodynamic importance. Here, this process is characterized using a simple analytical 'ball-and-spring' model and all-atom normal-mode analysis (NMA) of the binding of the cancer drug, methotrexate (MTX) to its target, dihydrofolate reductase (DHFR). The analytical model predicts that the coupling between protein vibrations and ligand external motion generates entropy-rich, low-frequency vibrations in the complex. This is consistent with the atomistic NMA which reveals vibrational softening in forming the DHFR-MTX complex, a result also in qualitative agreement with neutron-scattering experiments. Energy minimization of the atomistic bound-state (B) structure whilemore » gradually decreasing the ligand interaction to zero allows the generation of a hypothetical 'intermediate' (I) state, without the ligand force field but with a structure similar to that of B. In going from I to B, it is found that the vibrational entropies of both the protein and MTX decrease while the complex structure becomes enthalpically stabilized. However, the relatively weak DHFR:MTX interaction energy results in the net entropy gain arising from coupling between the protein and MTX external motion being larger than the loss of vibrational entropy on complex formation. This, together with the I structure being more flexible than the unbound structure, results in the observed vibrational softening on ligand binding.« less

Investigations on the Interactions of 5-Fluorouracil with Herring Sperm DNA: Steady State/Time Resolved and Molecular Modeling Studies

NASA Astrophysics Data System (ADS)

Chinnathambi, Shanmugavel; Karthikeyan, Subramani; Velmurugan, Devadasan; Hanagata, Nobutaka; Aruna, Prakasarao; Ganesan, Singaravelu

2015-04-01

In the present study, the interaction of 5-Fluorouracil with herring sperm DNA is reported using spectroscopic and molecular modeling techniques. This binding study of 5-FU with hs-DNA is of paramount importance in understanding chemico-biological interactions for drug design, pharmacy and biochemistry without altering the original structure. The challenge of the study was to find the exact binding mode of the drug 5-Fluorouracil with hs-DNA. From the absorption studies, a hyperchromic effect was observed for the herring sperm DNA in the presence of 5-Fluorouracil and a binding constant of 6.153 × 103 M-1 for 5-Fluorouracil reveals the existence of weak interaction between the 5-Fluorouracil and herring sperm DNA. Ethidium bromide loaded herring sperm DNA showed a quenching in the fluorescence intensity after the addition of 5-Fluorouracil. The binding constants for 5-Fluorouracil stranded DNA and competitive bindings of 5-FU interacting with DNA-EB systems were examined by fluorescence spectra. The Stern-Volmer plots and fluorescence lifetime results confirm the static quenching nature of the drug-DNA complex. The binding constant Kb was 2.5 × 104 L mol-1 and the number of binding sites are 1.17. The 5-FU on DNA system was calculated using double logarithmic plot. From the Forster nonradiative energy transfer study it has been found that the distance of 5-FU from DNA was 4.24 nm. In addition to the spectroscopic results, the molecular modeling studies also revealed the major groove binding as well as the partial intercalation mode of binding between the 5-Fluorouracil and herring sperm DNA. The binding energy and major groove binding as -6.04 kcal mol-1 and -6.31 kcal mol-1 were calculated from the modeling studies. All the testimonies manifested that binding modes between 5-Fluorouracil and DNA were evidenced to be groove binding and in partial intercalative mode.

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT).

PubMed

Yang, Hongfang; Medeiros, Patricia F; Raha, Kaushik; Elkins, Patricia; Lind, Kenneth E; Lehr, Ruth; Adams, Nicholas D; Burgess, Joelle L; Schmidt, Stanley J; Knight, Steven D; Auger, Kurt R; Schaber, Michael D; Franklin, G Joseph; Ding, Yun; DeLorey, Jennifer L; Centrella, Paolo A; Mataruse, Sibongile; Skinner, Steven R; Clark, Matthew A; Cuozzo, John W; Evindar, Ghotas

2015-05-14

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein.

Discovery of a Potent Class of PI3Kα Inhibitors with Unique Binding Mode via Encoded Library Technology (ELT)

PubMed Central

2015-01-01

In the search of PI3K p110α wild type and H1047R mutant selective small molecule leads, an encoded library technology (ELT) campaign against the desired target proteins was performed which led to the discovery of a selective chemotype for PI3K isoforms from a three-cycle DNA encoded library. An X-ray crystal structure of a representative inhibitor from this chemotype demonstrated a unique binding mode in the p110α protein. PMID:26005528

Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor

PubMed Central

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jurgen; Kobilka, Brian

2012-01-01

Acetylcholine (ACh), the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G protein-coupled receptors (GPCRs) known as muscarinic ACh receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G protein coupling preference and the physiological responses they mediate.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors. PMID:22358844

Structure and dynamics of the M3 muscarinic acetylcholine receptor

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

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.

2012-03-01

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify themore » binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.« less

The mode of inhibitor binding to peptidyl-tRNA hydrolase: binding studies and structure determination of unbound and bound peptidyl-tRNA hydrolase from Acinetobacter baumannii.

PubMed

Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2013-01-01

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.

Application of binding free energy calculations to prediction of binding modes and affinities of MDM2 and MDMX inhibitors.

PubMed

Lee, Hui Sun; Jo, Sunhwan; Lim, Hyun-Suk; Im, Wonpil

2012-07-23

Molecular docking is widely used to obtain binding modes and binding affinities of a molecule to a given target protein. Despite considerable efforts, however, prediction of both properties by docking remains challenging mainly due to protein's structural flexibility and inaccuracy of scoring functions. Here, an integrated approach has been developed to improve the accuracy of binding mode and affinity prediction and tested for small molecule MDM2 and MDMX antagonists. In this approach, initial candidate models selected from docking are subjected to equilibration MD simulations to further filter the models. Free energy perturbation molecular dynamics (FEP/MD) simulations are then applied to the filtered ligand models to enhance the ability in predicting the near-native ligand conformation. The calculated binding free energies for MDM2 complexes are overestimated compared to experimental measurements mainly due to the difficulties in sampling highly flexible apo-MDM2. Nonetheless, the FEP/MD binding free energy calculations are more promising for discriminating binders from nonbinders than docking scores. In particular, the comparison between the MDM2 and MDMX results suggests that apo-MDMX has lower flexibility than apo-MDM2. In addition, the FEP/MD calculations provide detailed information on the different energetic contributions to ligand binding, leading to a better understanding of the sensitivity and specificity of protein-ligand interactions.

Interaction of sucralose with whey protein: Experimental and molecular modeling studies

NASA Astrophysics Data System (ADS)

Zhang, Hongmei; Sun, Shixin; Wang, Yanqing; Cao, Jian

2017-12-01

The objective of this research was to study the interactions of sucralose with whey protein isolate (WPI) by using the three-dimensional fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling. The results showed that the peptide strands structure of WPI had been changed by sucralose. Sucralose binding induced the secondary structural changes and increased content of aperiodic structure of WPI. Sucralose decreased the thermal stability of WPI and acted as a structure destabilizer during the thermal unfolding process of protein. In addition, the existence of sucralose decreased the reversibility of the unfolding of WPI. Nonetheless, sucralose-WPI complex was less stable than protein alone. The molecular modeling result showed that van der Waals and hydrogen bonding interactions contribute to the complexation free binding energy. There are more than one possible binding sites of WPI with sucralose by surface binding mode.

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control.

PubMed

Satoh, Tadashi; Toshimori, Takayasu; Noda, Masanori; Uchiyama, Susumu; Kato, Koichi

2016-11-01

The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. © 2016 The Protein Society.

Structure-Activity Relationships of Small Molecule Autotaxin Inhibitors with a Discrete Binding Mode.

PubMed

Miller, Lisa M; Keune, Willem-Jan; Castagna, Diana; Young, Louise C; Duffy, Emma L; Potjewyd, Frances; Salgado-Polo, Fernando; Engel García, Paloma; Semaan, Dima; Pritchard, John M; Perrakis, Anastassis; Macdonald, Simon J F; Jamieson, Craig; Watson, Allan J B

2017-01-26

Autotaxin (ATX) is a secreted enzyme responsible for the hydrolysis of lysophosphatidylcholine (LPC) to the bioactive lysophosphatidic acid (LPA) and choline. The ATX-LPA signaling pathway is implicated in cell survival, migration, and proliferation; thus, the inhibition of ATX is a recognized therapeutic target for a number of diseases including fibrotic diseases, cancer, and inflammation, among others. Many of the developed synthetic inhibitors for ATX have resembled the lipid chemotype of the native ligand; however, a small number of inhibitors have been described that deviate from this common scaffold. Herein, we report the structure-activity relationships (SAR) of a previously reported small molecule ATX inhibitor. We show through enzyme kinetics studies that analogues of this chemotype are noncompetitive inhibitors, and by using a crystal structure with ATX we confirm the discrete binding mode.

Synthesis, characterization, crystal structure, DNA/BSA binding ability and antibacterial activity of asymmetric europium complex based on 1,10- phenanthroline

NASA Astrophysics Data System (ADS)

Alfi, Nafiseh; Khorasani-Motlagh, Mozhgan; Rezvani, Ali Reza; Noroozifar, Meissam; Molčanov, Krešimir

2017-06-01

A heteroleptic europium coordination compound formulated as [Eu(phen)2(OH2)2(Cl)2](Cl)(H2O) (phen = 1,10-phenanthroline), has been synthesized and characterized by elemental analysis, FT-IR spectroscopy, and single-crystal X-ray diffractometer. Crystal structure analysis reveals the complex is crystallized in orthorhombic system with Pca21 space group. Electronic absorption and various emission methods for investigation of the binding system of europium(III) complex to Fish Salmon deoxyribonucleic acid (FS-DNA) and Bovamin Serum Albumin (BSA) have been explored. Furthermore, the binding constants, binding sites and the corresponding thermodynamic parameters of the interaction system based on the van't Hoff equation for FS-DNA and BSA were calculated. The thermodynamic parameters reflect the exothermic nature of emission process (ΔH°<0 and ΔS°<0). The experimental results seem to indicate that the [Eu(phen)2(OH2)2(Cl)2](Cl)(H2O) bound to FS-DNA by non-intercalative mode which the groove binding is preferable mode. Also, the complex exhibits a brilliant antimicrobial activity in vitro against standard bacterial strains.

Membrane interaction and functional plasticity of inositol polyphosphate 5-phosphatases.

PubMed

Braun, Werner; Schein, Catherine H

2014-05-06

In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center. Copyright © 2014 Elsevier Ltd. All rights reserved.

Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A

PubMed Central

Kaminishi, Tatsuya; Schedlbauer, Andreas; Fabbretti, Attilio; Brandi, Letizia; Ochoa-Lizarralde, Borja; He, Cheng-Guang; Milón, Pohl; Connell, Sean R.; Gualerzi, Claudio O.; Fucini, Paola

2015-01-01

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. PMID:26464437

Thermodynamics and binding mode of novel structurally related 1,2,4-thiadiazole derivatives with native and modified cyclodextrins

NASA Astrophysics Data System (ADS)

Terekhova, Irina V.; Chislov, Mikhail V.; Brusnikina, Maria A.; Chibunova, Ekaterina S.; Volkova, Tatyana V.; Zvereva, Irina A.; Proshin, Alexey N.

2017-03-01

Study of complex formation of cyclodextrins with 1,2,4-thiadiazole derivatives intended for Alzheimer's disease treatment was carried out using 1H NMR, ITC and phase solubility methods. Structure of cyclodextrins and thiadiazoles affects the binding mode and thermodynamics of complexation. The larger cavity of β- and γ-cyclodextrins is more appropriate for deeper insertion of 1,2,4-thiadiazole derivatives which is accompanied by intensive dehydration and solvent reorganization. Benzene ring of the thiadiazoles is located inside macrocyclic cavity while piperidine ring is placed outside the cavity and can form H-bonds with cyclodextrin exterior. Complexation with cyclodextrins induces the enhancement of aqueous solubility of 1,2,4-thiadiazole derivatives.

Unusual mode of protein binding by a cytotoxic π-arene ruthenium(ii) piano-stool compound containing an O,S-chelating ligand.

PubMed

Hildebrandt, Jana; Görls, Helmar; Häfner, Norman; Ferraro, Giarita; Dürst, Matthias; Runnebaum, Ingo B; Weigand, Wolfgang; Merlino, Antonello

2016-08-02

A new pseudo-octahedral π-arene ruthenium(ii) piano-stool compound, containing an O,S-bidentate ligand (compound 1) and showing significant cytotoxic activity in vitro, was synthesized and characterized. In solution stability and interaction with the model protein bovine pancreatic ribonuclease (RNase A) were investigated by using UV-Vis absorption spectroscopy. Its crystal structure and that of the adduct formed upon reaction with RNase A were obtained by X-ray crystallography. The comparison between the structure of purified compound 1 and that of the fragment bound to RNase A reveals an unusual mode of protein binding that includes ligand exchange and alteration of coordination sphere geometry.

Exploring the stability of ligand binding modes to proteins by molecular dynamics simulations.

PubMed

Liu, Kai; Watanabe, Etsurou; Kokubo, Hironori

2017-02-01

The binding mode prediction is of great importance to structure-based drug design. The discrimination of various binding poses of ligand generated by docking is a great challenge not only to docking score functions but also to the relatively expensive free energy calculation methods. Here we systematically analyzed the stability of various ligand poses under molecular dynamics (MD) simulation. First, a data set of 120 complexes was built based on the typical physicochemical properties of drug-like ligands. Three potential binding poses (one correct pose and two decoys) were selected for each ligand from self-docking in addition to the experimental pose. Then, five independent MD simulations for each pose were performed with different initial velocities for the statistical analysis. Finally, the stabilities of ligand poses under MD were evaluated and compared with the native one from crystal structure. We found that about 94% of the native poses were maintained stable during the simulations, which suggests that MD simulations are accurate enough to judge most experimental binding poses as stable properly. Interestingly, incorrect decoy poses were maintained much less and 38-44% of decoys could be excluded just by performing equilibrium MD simulations, though 56-62% of decoys were stable. The computationally-heavy binding free energy calculation can be performed only for these survived poses.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

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

2014-01-01

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

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.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

Binding Modes of Ligands Using Enhanced Sampling (BLUES): Rapid Decorrelation of Ligand Binding Modes via Nonequilibrium Candidate Monte Carlo.

PubMed

Gill, Samuel C; Lim, Nathan M; Grinaway, Patrick B; Rustenburg, Ariën S; Fass, Josh; Ross, Gregory A; Chodera, John D; Mobley, David L

2018-05-31

Accurately predicting protein-ligand binding affinities and binding modes is a major goal in computational chemistry, but even the prediction of ligand binding modes in proteins poses major challenges. Here, we focus on solving the binding mode prediction problem for rigid fragments. That is, we focus on computing the dominant placement, conformation, and orientations of a relatively rigid, fragment-like ligand in a receptor, and the populations of the multiple binding modes which may be relevant. This problem is important in its own right, but is even more timely given the recent success of alchemical free energy calculations. Alchemical calculations are increasingly used to predict binding free energies of ligands to receptors. However, the accuracy of these calculations is dependent on proper sampling of the relevant ligand binding modes. Unfortunately, ligand binding modes may often be uncertain, hard to predict, and/or slow to interconvert on simulation time scales, so proper sampling with current techniques can require prohibitively long simulations. We need new methods which dramatically improve sampling of ligand binding modes. Here, we develop and apply a nonequilibrium candidate Monte Carlo (NCMC) method to improve sampling of ligand binding modes. In this technique, the ligand is rotated and subsequently allowed to relax in its new position through alchemical perturbation before accepting or rejecting the rotation and relaxation as a nonequilibrium Monte Carlo move. When applied to a T4 lysozyme model binding system, this NCMC method shows over 2 orders of magnitude improvement in binding mode sampling efficiency compared to a brute force molecular dynamics simulation. This is a first step toward applying this methodology to pharmaceutically relevant binding of fragments and, eventually, drug-like molecules. We are making this approach available via our new Binding modes of ligands using enhanced sampling (BLUES) package which is freely available on GitHub.

Probing the binding of flavonoids to catalase by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Zhu, Jingfeng; Zhang, Xia; Li, Daojin; Jin, Jing

2007-10-01

The binding of flavonoids (quercetin and myricetin) to catalase was investigated by fluorescence and circular dichroism (CD) techniques under physiological conditions. The binding parameters and binding mode between flavonoids and catalase were determined, and the results of synchronous fluorescence spectra and CD indicated a conformational change of catalase with addition of flavonoids. The effect of both Cu 2+ and vitamin C on the binding constant of flavonoid-catalase was also examined. The experiment data show that the difference of the structure characteristics of quercetin and myricetin has a significant effect on their binding affinity for catalase.

Stiffening of flexible SUMO1 protein upon peptide-binding: Analysis with anisotropic network model.

PubMed

Sarkar, Ranja

2018-01-01

SUMO (small ubiquitin-like modifier) proteins interact with a large number of target proteins via a key regulatory event called sumoylation that encompasses activation, conjugation and ligation of SUMO proteins through specific E1, E2, and E3-type enzymes respectively. Single-molecule atomic force microscopic (AFM) experiments performed to unravel bound SUMO1 along its NC termini direction reveal that E3-ligases (in the form of small peptides) increase mechanical stability (along the axis) of the flexible protein upon binding. The experimental results are expected to correlate with the intrinsic flexibility of bound SUMO1 protein in the native state i.e., the bound conformation of SUMO1 without the binding peptide. The native protein flexibility/stiffness can be measured as a spring constant by normal mode analysis. In the present study, protein normal modes are computed from the protein structural data (as input from protein databank) via a simple anisotropic network model (ANM). ANM is computationally inexpensive and hence, can be explored to investigate and compare the native conformational dynamics of unbound and bound (without the binding partner) structures, if the corresponding structural data (NMR/X-ray) are available. The paper illustrates that SUMO1 stiffens (native flexibility decreases) along the NC termini (end-to-end) direction of the protein upon binding to small peptides; however, the degree of stiffening is peptide sequence-specific. The theoretical results are demonstrated for NMR structures of unbound SUMO1 and that bound to two peptides having short amino acid motifs and of similar size, one being an M-IR2 peptide derived from RanBP2 protein and the other one derived from PIASX protein. The peptide derived from PIASX stiffens SUMO1 remarkably which is evident from an atomic-level normal mode analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations

NASA Astrophysics Data System (ADS)

Martins, Luan Carvalho; Torres, Pedro Henrique Monteiro; de Oliveira, Renata Barbosa; Pascutti, Pedro Geraldo; Cino, Elio A.; Ferreira, Rafaela Salgado

2018-05-01

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations

NASA Astrophysics Data System (ADS)

Martins, Luan Carvalho; Torres, Pedro Henrique Monteiro; de Oliveira, Renata Barbosa; Pascutti, Pedro Geraldo; Cino, Elio A.; Ferreira, Rafaela Salgado

2018-03-01

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations.

PubMed

Martins, Luan Carvalho; Torres, Pedro Henrique Monteiro; de Oliveira, Renata Barbosa; Pascutti, Pedro Geraldo; Cino, Elio A; Ferreira, Rafaela Salgado

2018-05-01

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC 50  = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

Design and Synthesis of a Pan-Janus Kinase Inhibitor Clinical Candidate (PF-06263276) Suitable for Inhaled and Topical Delivery for the Treatment of Inflammatory Diseases of the Lungs and Skin

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

Jones, Peter; Storer, R. Ian; Sabnis, Yogesh A.

By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

PubMed

Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

2013-02-15

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Exploring the Inhibitory Mechanism of Approved Selective Norepinephrine Reuptake Inhibitors and Reboxetine Enantiomers by Molecular Dynamics Study

NASA Astrophysics Data System (ADS)

Zheng, Guoxun; Xue, Weiwei; Wang, Panpan; Yang, Fengyuan; Li, Bo; Li, Xiaofeng; Li, Yinghong; Yao, Xiaojun; Zhu, Feng

2016-05-01

Selective norepinephrine reuptake inhibitors (sNRIs) provide an effective class of approved antipsychotics, whose inhibitory mechanism could facilitate the discovery of privileged scaffolds with enhanced drug efficacy. However, the crystal structure of human norepinephrine transporter (hNET) has not been determined yet and the inhibitory mechanism of sNRIs remains elusive. In this work, multiple computational methods were integrated to explore the inhibitory mechanism of approved sNRIs (atomoxetine, maprotiline, reboxetine and viloxazine), and 3 lines of evidences were provided to verify the calculation results. Consequently, a binding mode defined by interactions between three chemical moieties in sNRIs and eleven residues in hNET was identified as shared by approved sNRIs. In the meantime, binding modes of reboxetine’s enantiomers with hNET were compared. 6 key residues favoring the binding of (S, S)-reboxetine over that of (R, R)-reboxetine were discovered. This is the first study reporting that those 11 residues are the common determinants for the binding of approved sNRIs. The identified binding mode shed light on the inhibitory mechanism of approved sNRIs, which could help identify novel scaffolds with improved drug efficacy.

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

PubMed

Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-10-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host

PubMed Central

Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-01-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

PubMed Central

Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

2010-01-01

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

Thermal perturbation correlation of calcium binding Human centrin 3 and its structural changes

NASA Astrophysics Data System (ADS)

Pastrana-Rios, Belinda

2014-07-01

Perturbation-correlation moving-window two-dimensional (PCMW2D) correlation spectroscopy was applied for the determination of the individual transition temperatures of different vibrational modes located within structural components of a calcium binding protein known as Human centrin 3. This crucial information served to understand the contribution individual calcium binding sites made towards the stability of the EF-hand and therefore the protein without the use of probes. We are convinced that the general application of PCMW2D correlation spectroscopy can be applied to the study of proteins in general to ascertain the differences in the stability of structural motifs within proteins and its relationship to the actual transition temperature of unfolding.

A small-molecule fragment that emulates binding of receptor and broadly neutralizing antibodies to influenza A hemagglutinin.

PubMed

Kadam, Rameshwar U; Wilson, Ian A

2018-04-17

The influenza virus hemagglutinin (HA) glycoprotein mediates receptor binding and membrane fusion during viral entry in host cells. Blocking these key steps in viral infection has applications for development of novel antiinfluenza therapeutics as well as vaccines. However, the lack of structural information on how small molecules can gain a foothold in the small, shallow receptor-binding site (RBS) has hindered drug design against this important target on the viral pathogen. Here, we report on the serendipitous crystallization-based discovery of a small-molecule N -cyclohexyltaurine, commonly known as the buffering agent CHES, that is able to bind to both group-1 and group-2 HAs of influenza A viruses. X-ray structural characterization of group-1 H5N1 A/Vietnam/1203/2004 (H5/Viet) and group-2 H3N2 A/Hong Kong/1/1968 (H3/HK68) HAs at 2.0-Å and 2.57-Å resolution, respectively, revealed that N -cyclohexyltaurine binds to the heart of the conserved HA RBS. N -cyclohexyltaurine mimics the binding mode of the natural receptor sialic acid and RBS-targeting bnAbs through formation of similar hydrogen bonds and CH-π interactions with the HA. In H3/HK68, N -cyclohexyltaurine also binds to a conserved pocket in the stem region, thereby exhibiting a dual-binding mode in group-2 HAs. These long-awaited structural insights into RBS recognition by a noncarbohydrate-based small molecule enhance our knowledge of how to target this important functional site and can serve as a template to guide the development of novel broad-spectrum small-molecule therapeutics against influenza virus.

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein.

PubMed

Martino, Luigi; Pennell, Simon; Kelly, Geoff; Bui, Tam T T; Kotik-Kogan, Olga; Smerdon, Stephen J; Drake, Alex F; Curry, Stephen; Conte, Maria R

2012-02-01

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3' UUU(OH) trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3' oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.

p53 Specifically Binds Triplex DNA In Vitro and in Cells

PubMed Central

Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L.; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

2016-01-01

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed. PMID:27907175

Synthesis, spectroscopic characterization, DNA interaction and biological activities of Mn(II), Co(II), Ni(II) and Cu(II) complexes with [(1H-1,2,4-triazole-3-ylimino)methyl]naphthalene-2-ol

NASA Astrophysics Data System (ADS)

Gaber, Mohamed; El-Wakiel, Nadia A.; El-Ghamry, Hoda; Fathalla, Shaimaa K.

2014-11-01

Manganese(II), cobalt(II), nickel(II) and copper(II) complexes of [(1H-1,2,4-triazole-3-ylimino)methyl]naphthalene-2-ol have been synthesized. The structure of complexes have been characterized by elemental analysis, molar conductance, magnetic moment measurements and spectral (IR, 1H NMR, EI-mass, UV-Vis and ESR), and thermal studies. The results showed that the chloro and nitrato Cu(II) complexes have octahedral geometry while Ni(II), Co(II) and Mn(II) complexes in addition to acetato Cu(II) complex have tetrahedral geometry. The possible structures of the metal complexes have been computed using the molecular mechanic calculations using the hyper chem. 8.03 molecular modeling program to confirm the proposed structures. The kinetic and thermodynamic parameters of the thermal decomposition steps were calculated from the TG curves. The binding modes of the complexes with DNA have been investigated by UV-Vis absorption titration. The results showed that the mode of binding of the complexes to DNA is intercalative or non-intercalative binding modes. Schiff base and its metal complexes have been screened for their in vitro antimicrobial activities against Gram positive bacteria (Staphylococcus aureus), Gram negative bacteria (Escherichia coli and Pesudomonas aeruginosa), fungi (Asperigllus flavus and Mucer) and yeast (Candida albicans and Malassezia furfur).

Molecular mechanism of peroxisome proliferator-activated receptor α activation by WY14643: a new mode of ligand recognition and receptor stabilization.

PubMed

Bernardes, Amanda; Souza, Paulo C T; Muniz, João R C; Ricci, Clarisse G; Ayers, Stephen D; Parekh, Nili M; Godoy, André S; Trivella, Daniela B B; Reinach, Peter; Webb, Paul; Skaf, Munir S; Polikarpov, Igor

2013-08-23

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands. Copyright © 2013 Elsevier Ltd. All rights reserved.

Conformational dynamics and ligand binding in the multi-domain protein PDC109.

PubMed

Kim, Hyun Jin; Choi, Moo Young; Kim, Hyung J; Llinás, Miguel

2010-02-18

PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2) repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs), a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD) simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M(-1), estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity

DOE PAGES

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; ...

2015-01-09

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD +, which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexesmore » with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD + and XMP/NAD +. In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD+ adenosine moiety. More importantly, this new NAD +-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD +-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. As a result, these findings offer a potential strategy for further ligand optimization.« less

A novel cofactor-binding mode in bacterial IMP dehydrogenases explains inhibitor selectivity.

PubMed

Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Osipiuk, Jerzy; Gu, Minyi; Zhang, Minjia; Mandapati, Kavitha; Gollapalli, Deviprasad R; Gorla, Suresh Kumar; Hedstrom, Lizbeth; Joachimiak, Andrzej

2015-02-27

The steadily rising frequency of emerging diseases and antibiotic resistance creates an urgent need for new drugs and targets. Inosine 5'-monophosphate dehydrogenase (IMP dehydrogenase or IMPDH) is a promising target for the development of new antimicrobial agents. IMPDH catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD(+), which is the pivotal step in the biosynthesis of guanine nucleotides. Potent inhibitors of bacterial IMPDHs have been identified that bind in a structurally distinct pocket that is absent in eukaryotic IMPDHs. The physiological role of this pocket was not understood. Here, we report the structures of complexes with different classes of inhibitors of Bacillus anthracis, Campylobacter jejuni, and Clostridium perfringens IMPDHs. These structures in combination with inhibition studies provide important insights into the interactions that modulate selectivity and potency. We also present two structures of the Vibrio cholerae IMPDH in complex with IMP/NAD(+) and XMP/NAD(+). In both structures, the cofactor assumes a dramatically different conformation than reported previously for eukaryotic IMPDHs and other dehydrogenases, with the major change observed for the position of the NAD(+) adenosine moiety. More importantly, this new NAD(+)-binding site involves the same pocket that is utilized by the inhibitors. Thus, the bacterial IMPDH-specific NAD(+)-binding mode helps to rationalize the conformation adopted by several classes of prokaryotic IMPDH inhibitors. These findings offer a potential strategy for further ligand optimization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

DFT based vibrational spectroscopic investigations and biological activity of toxic material monocrotophos

NASA Astrophysics Data System (ADS)

Nimmi, D. E.; Sam, S. P. Chandhini; Praveen, S. G.; Binoy, J.

2018-05-01

Many organophosphate compounds exhibiting toxicity are widely used as pesticides and insecticides whose structural features can be explained excellently using geometric simulation using density functional theory and vibrational spectrum. In this work, the molecular structural parameters and vibrational frequencies of the fundamental modes of Monocrotophoshave been obtained using Density functional theory (DFT), using B3LYP functional with 6-311++G(d, p) basis sets and the detailed vibrational analysis of FT-IR and FT-Ramanspectral bands have been carried out using potential energy distribution (PED). The deviation from the resonance structure of phosphate group due to `bridging of oxygen' and π-resonance of amides has been investigated based on the spectral and geometric data. The molecular docking simulation of Monocrotophos with BSA and DNA has been performed to find the mode of binding and the interactions with BSA has been investigated with UV-Visible spectroscopic method, to assess the strength of binding.

Phosphate forms an unusual tripodal complex with the Fe–Mn center of sweet potato purple acid phosphatase

PubMed Central

Schenk, Gerhard; Gahan, Lawrence R.; Carrington, Lyle E.; Mitić, Nataša; Valizadeh, Mohsen; Hamilton, Susan E.; de Jersey, John; Guddat, Luke W.

2005-01-01

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)–Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a μ-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (kcat/Km) at pH 4.5, whereas its catalytic rate constant (kcat) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pKa of the leaving group. The crystal structure of the phosphate-bound Fe(III)–Mn(II) PAP has been determined to 2.5-Å resolution (final Rfree value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to λ protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis. PMID:15625111

Design of Broad-Spectrum Inhibitors of Influenza A Virus M2 Proton Channels: A Molecular Modeling Approach.

PubMed

Klimochkin, Yuri N; Shiryaev, Vadim A; Petrov, Pavel V; Radchenko, Eugene V; Palyulin, Vladimir A; Zefirov, Nikolay S

2016-01-01

The influenza A virus M2 proton channel plays a critical role in its life cycle. However, known M2 inhibitors have lost their clinical efficacy due to the spread of resistant mutant channels. Thus, the search for broad-spectrum M2 channel inhibitors is of great importance. The goal of the present work was to develop a general approach supporting the design of ligands interacting with multiple labile targets and to propose on its basis the potential broad-spectrum inhibitors of the M2 proton channel. The dynamic dimer-of-dimers structures of the three primary M2 target variants, wild-type, S31N and V27A, were modeled by molecular dynamics and thoroughly analyzed in order to define the inhibitor binding sites. The potential inhibitor structures were identified by molecular docking and their binding was verified by molecular dynamics simulation. The binding sites of the M2 proton channel inhibitors were analyzed, a number of potential broad-spectrum inhibitors were identified and the binding modes and probable mechanisms of action of one promising compound were clarified. Using the molecular dynamics and molecular docking techniques, we have refined the dynamic dimer-ofdimers structures of the WT, S31N and V27A variants of the M2 proton channel of the influenza A virus, analyzed the inhibitor binding sites, identified a number of potential broad-spectrum inhibitor structures targeting them, and clarified the binding modes and probable mechanisms of action of one promising compound. The proposed approach is also suitable for the design of ligands interacting with other multiple labile targets.

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

PubMed Central

Hoffmann, Andreas; Kovermann, Michael; Lilie, Hauke; Fiedler, Markus; Balbach, Jochen; Rudolph, Rainer; Pfeifer, Sven

2012-01-01

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies. PMID:22363609

Molecular simulations enlighten the binding mode of quercetin to lipoxygenase-3.

PubMed

Fiorucci, Sébastien; Golebiowski, Jérôme; Cabrol-Bass, Daniel; Antonczak, Serge

2008-11-01

Inhibition of lipoxygenases (LOXs) by flavonoid compounds is now well documented, but the description of the associated mechanism remains controversial due to a lack of information at the molecular level. For instance, X-ray determination of quercetin/LOX-3 system has led to a structure where the enzyme was cocrystallized with a degradation product of the substrate, which rendered the interpretation of the reported interactions between this flavonoid compound and the enzyme difficult. Molecular modeling simulations can in principle allow obtaining precious insights that could fill this lack of structural information. Thus, in this study, we have investigated various binding modes of quercetin to LOX-3 enzyme in order to understand the first step of the inhibition process, that is the association of the two entities. Molecular dynamics simulations and free energy calculations suggest that quercetin binds the metal center via its 3-hydroxychromone function. Moreover, enzyme/substrate interactions within the cavity impose steric hindrances to quercetin that may activate a direct dioxygen addition on the substrate. (c) 2008 Wiley-Liss, Inc.

Hit to lead account of the discovery of a new class of inhibitors of Pim kinases and crystallographic studies revealing an unusual kinase binding mode.

PubMed

Qian, Kevin; Wang, Lian; Cywin, Charles L; Farmer, Bennett T; Hickey, Eugene; Homon, Carol; Jakes, Scott; Kashem, Mohammed A; Lee, George; Leonard, Scott; Li, Jun; Magboo, Ronald; Mao, Wang; Pack, Edward; Peng, Charlene; Prokopowicz, Anthony; Welzel, Morgan; Wolak, John; Morwick, Tina

2009-04-09

A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 K(M(ATP)) values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural features which may contribute to the association are discussed.

CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

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

Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. Themore » KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.« less

Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

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

Shen, Aimee; Higgins, Darren E.; Panne, Daniel

2009-07-22

The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity.more » The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.« less

Binding Modes of Phthalocyanines to Amyloid β Peptide and Their Effects on Amyloid Fibril Formation.

PubMed

Valiente-Gabioud, Ariel A; Riedel, Dietmar; Outeiro, Tiago F; Menacho-Márquez, Mauricio A; Griesinger, Christian; Fernández, Claudio O

2018-03-13

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer's and Parkinson's diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid β-peptide (Aβ) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aβ 40 revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aβ 40 -PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aβ 40 -PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aβ 40 and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of pathological aggregates. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. 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.

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

Fold independent structural comparisons of protein-ligand binding sites for exploring functional relationships.

PubMed

Gold, Nicola D; Jackson, Richard M

2006-02-03

The rapid growth in protein structural data and the emergence of structural genomics projects have increased the need for automatic structure analysis and tools for function prediction. Small molecule recognition is critical to the function of many proteins; therefore, determination of ligand binding site similarity is important for understanding ligand interactions and may allow their functional classification. Here, we present a binding sites database (SitesBase) that given a known protein-ligand binding site allows rapid retrieval of other binding sites with similar structure independent of overall sequence or fold similarity. However, each match is also annotated with sequence similarity and fold information to aid interpretation of structure and functional similarity. Similarity in ligand binding sites can indicate common binding modes and recognition of similar molecules, allowing potential inference of function for an uncharacterised protein or providing additional evidence of common function where sequence or fold similarity is already known. Alternatively, the resource can provide valuable information for detailed studies of molecular recognition including structure-based ligand design and in understanding ligand cross-reactivity. Here, we show examples of atomic similarity between superfamily or more distant fold relatives as well as between seemingly unrelated proteins. Assignment of unclassified proteins to structural superfamiles is also undertaken and in most cases substantiates assignments made using sequence similarity. Correct assignment is also possible where sequence similarity fails to find significant matches, illustrating the potential use of binding site comparisons for newly determined proteins.

Rhodopsin TM6 Can Interact with Two Separate and Distinct Sites on Arrestin: Evidence for Structural Plasticity and Multiple Docking Modes in Arrestin–Rhodopsin Binding

PubMed Central

2015-01-01

Various studies have implicated the concave surface of arrestin in the binding of the cytosolic surface of rhodopsin. However, specific sites of contact between the two proteins have not previously been defined in detail. Here, we report that arrestin shares part of the same binding site on rhodopsin as does the transducin Gα subunit C-terminal tail, suggesting binding of both proteins to rhodopsin may share some similar underlying mechanisms. We also identify two areas of contact between the proteins near this region. Both sites lie in the arrestin N-domain, one in the so-called “finger” loop (residues 67–79) and the other in the 160 loop (residues 155–165). We mapped these sites using a novel tryptophan-induced quenching method, in which we introduced Trp residues into arrestin and measured their ability to quench the fluorescence of bimane probes attached to cysteine residues on TM6 of rhodopsin (T242C and T243C). The involvement of finger loop binding to rhodopsin was expected, but the evidence of the arrestin 160 loop contacting rhodopsin was not. Remarkably, our data indicate one site on rhodopsin can interact with multiple structurally separate sites on arrestin that are almost 30 Å apart. Although this observation at first seems paradoxical, in fact, it provides strong support for recent hypotheses that structural plasticity and conformational changes are involved in the arrestin–rhodopsin binding interface and that the two proteins may be able to interact through multiple docking modes, with arrestin binding to both monomeric and dimeric rhodopsin. PMID:24724832

Rhodopsin TM6 can interact with two separate and distinct sites on arrestin: evidence for structural plasticity and multiple docking modes in arrestin-rhodopsin binding.

PubMed

Sinha, Abhinav; Jones Brunette, Amber M; Fay, Jonathan F; Schafer, Christopher T; Farrens, David L

2014-05-27

Various studies have implicated the concave surface of arrestin in the binding of the cytosolic surface of rhodopsin. However, specific sites of contact between the two proteins have not previously been defined in detail. Here, we report that arrestin shares part of the same binding site on rhodopsin as does the transducin Gα subunit C-terminal tail, suggesting binding of both proteins to rhodopsin may share some similar underlying mechanisms. We also identify two areas of contact between the proteins near this region. Both sites lie in the arrestin N-domain, one in the so-called "finger" loop (residues 67-79) and the other in the 160 loop (residues 155-165). We mapped these sites using a novel tryptophan-induced quenching method, in which we introduced Trp residues into arrestin and measured their ability to quench the fluorescence of bimane probes attached to cysteine residues on TM6 of rhodopsin (T242C and T243C). The involvement of finger loop binding to rhodopsin was expected, but the evidence of the arrestin 160 loop contacting rhodopsin was not. Remarkably, our data indicate one site on rhodopsin can interact with multiple structurally separate sites on arrestin that are almost 30 Å apart. Although this observation at first seems paradoxical, in fact, it provides strong support for recent hypotheses that structural plasticity and conformational changes are involved in the arrestin-rhodopsin binding interface and that the two proteins may be able to interact through multiple docking modes, with arrestin binding to both monomeric and dimeric rhodopsin.

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs)

PubMed Central

Bayfield, Mark A.; Yang, Ruiqing; Maraia, Richard J.

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3’OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3’OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3’OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNA assembly by hLARP7/PIP7S). Analyses of other LARP family members (i.e., hLARP4, hLARP6) suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs. PMID:20138158

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs).

PubMed

Bayfield, Mark A; Yang, Ruiqing; Maraia, Richard J

2010-01-01

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3'OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La-RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3'OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3'OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNP assembly by hLARP7/PIP7S). Analyses of other LARP family members suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA-related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs.

A Blocking Group Scan Using a Spherical Organometallic Complex Identifies an Unprecedented Binding Mode with Potent Activity In Vitro and In Vivo for the Opioid Peptide Dermorphin.

PubMed

Strack, Martin; Bedini, Andrea; Yip, King T; Lombardi, Sara; Siegmund, Daniel; Stoll, Raphael; Spampinato, Santi M; Metzler-Nolte, Nils

2016-10-04

Herein, the selective enforcement of one particular receptor-ligand interaction between specific domains of the μ-selective opioid peptide dermorphin and the μ opioid receptor is presented. For this, a blocking group scan is described which exploits the steric demand of a bis(quinolinylmethyl)amine rhenium(I) tricarbonyl complex conjugated to a number of different, strategically chosen positions of dermorphin. The prepared peptide conjugates lead to the discovery of two different binding modes: An expected N-terminal binding mode corresponds to the established view of opioid peptide binding, whereas an unexpected C-terminal binding mode is newly discovered. Surprisingly, both binding modes provide high affinity and agonistic activity at the μ opioid receptor in vitro. Furthermore, the unprecedented C-terminal binding mode shows potent dose-dependent antinociception in vivo. Finally, in silico docking studies support receptor activation by both dermorphin binding modes and suggest a biological relevance for dermorphin itself. Relevant ligand-protein interactions are similar for both binding modes, which is in line with previous protein mutation studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Picosecond dynamics of photoexcited DNO-bound myoglobin probed by femtosecond vibrational spectroscopy.

PubMed

Lee, Taegon; Hwang, Sungu; Lim, Manho

2015-02-05

Like nitric oxide (NO), nitroxyl (HNO), a reduced form of NO, plays many biologically important roles including neurological function and vascular regulation. Although HNO is unstable in aqueous solution, it is exceptionally stable on binding to ferrous myoglobin (Mb) to form MbHNO. Various experimental and theoretical investigations has been carried out to unveil the structure of the active site and binding characteristics of MbHNO that can explain its functioning mechanism and the origin of its unusual stability. However, the binding dynamics of HNO to Mb, as well as the photochemical and photophysical processes associated with binding, have not been fully established. Herein, femtosecond vibrational spectroscopy was used to probe the photoexcitation dynamics of excited MbDNO in D2O solution at 294 K with a 575 nm pulse. Time-resolved spectra were described by three vibrational bands near 1380 cm(-1), in the expected N-O stretching (νN-O) mode of MbDNO, and all three bands showed instantaneous bleach that decays on a picosecond time scale. The three bands were assigned based on isotope shifts upon (15)N substitution and ab initio calculation of the vibrational frequency on a DNO-bound model heme. These three bands likely arise from Fermi interactions between the strong νN-O mode and the weak overtone and combination modes of the N atom-related modes. The immediate appearance of the bleach in these bands and the picosecond decay of the bleach indicate that most of the photoexcited MbDNO undergoes picosecond geminate rebinding (GR) of DNO to Mb subsequent to its immediate deligation. Ultrafast and efficient GR of DNO likely arises from the bonding structure of the ligand and high reactivity between DNO and Mb.

Structures of the APC–ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners

PubMed Central

Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

2015-01-01

The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC–Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC–ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC–ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC–ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC–ARM binding partners. PMID:27462415

Structures of the APC-ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners.

PubMed

Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

2015-01-01

The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC-Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC-ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC-ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC-ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC-ARM binding partners.

Investigation of antibacterial mode of action for traditional and amphiphilic aminoglycosides.

PubMed

Udumula, Venkatareddy; Ham, Young Wan; Fosso, Marina Y; Chan, Ka Yee; Rai, Ravi; Zhang, Jianjun; Li, Jie; Chang, Cheng-Wei Tom

2013-03-15

Aminoglycoside represents a class of versatile and broad spectrum antibacterial agents. In an effort to revive the antibacterial activity against aminoglycoside resistant bacteria, our laboratory has developed two new classes of aminoglycoside, pyranmycin and amphiphilic neomycin (NEOF004). The former resembles the traditional aminoglycoside, neomycin. The latter, albeit derived from neomycin, appears to exert antibacterial action via a different mode of action. In order to discern that these aminoglycoside derivatives have distinct antibacterial mode of action, RNA-binding affinity and fluorogenic dye were employed. These studies, together with our previous investigation, confirm that pyranmycin exhibit the traditional antibacterial mode of action of aminoglycosides by binding toward the bacterial rRNA. On the other hand, the amphiphilic neomycin, NEOF004 disrupts the bacterial cell wall. In a broader perspective, it verifies that structurally modified neomycin can exert different antibacterial mode of action leading to the revival of activity against aminoglycoside resistant bacteria. Copyright © 2013 Elsevier Ltd. All rights reserved.

Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

PubMed

Jeřábek, Petr; Florián, Jan; Stiborová, Marie; Martínek, Václav

2014-10-28

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

Nature of Interlayer Binding and Stacking of sp–sp 2 Hybridized Carbon Layers: A Quantum Monte Carlo Study

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

Shin, Hyeondeok; Kim, Jeongnim; Lee, Hoonkyung

α-graphyne is a two-dimensional sheet of sp-sp2 hybridized carbon atoms in a honeycomb lattice. While the geometrical structure is similar to that of graphene, the hybridized triple bonds give rise to electronic structure that is different from that of graphene. Similar to graphene, α-graphyne can be stacked in bilayers with two stable configurations, but the different stackings have very different electronic structures: one is predicted to have gapless parabolic bands and the other a tunable bandgap which is attractive for applications. In order to realize applications, it is crucial to understand which stacking is more stable. This is difficult tomore » model, as the stability is a result of weak interlayer van der Waals interactions which are not well captured by density functional theory (DFT). We have used quantum Monte Carlo simulations that accurately include van der Waals interactions to calculate the interlayer binding energy of bilayer graphyne and to determine its most stable stacking mode. Our results show that inter-layer bindings of sp- and sp2-bonded carbon networks are significantly underestimated in a Kohn-Sham DFT approach, even with an exchange-correlation potential corrected to include, in some approximation, van der Waals interactions. Finally, our quantum Monte Carlo calculations reveal that the interlayer binding energy difference between the two stacking modes is only 0.9(4) eV/atom. From this we conclude that the two stable stacking modes of bilayer α-graphyne are almost degenerate with each other, and both will occur with about the same probability at room temperature unless there is a synthesis path that prefers one stacking over the other.« less

Nature of Interlayer Binding and Stacking of sp–sp 2 Hybridized Carbon Layers: A Quantum Monte Carlo Study

DOE PAGES

Shin, Hyeondeok; Kim, Jeongnim; Lee, Hoonkyung; ...

2017-10-25

α-graphyne is a two-dimensional sheet of sp-sp2 hybridized carbon atoms in a honeycomb lattice. While the geometrical structure is similar to that of graphene, the hybridized triple bonds give rise to electronic structure that is different from that of graphene. Similar to graphene, α-graphyne can be stacked in bilayers with two stable configurations, but the different stackings have very different electronic structures: one is predicted to have gapless parabolic bands and the other a tunable bandgap which is attractive for applications. In order to realize applications, it is crucial to understand which stacking is more stable. This is difficult tomore » model, as the stability is a result of weak interlayer van der Waals interactions which are not well captured by density functional theory (DFT). We have used quantum Monte Carlo simulations that accurately include van der Waals interactions to calculate the interlayer binding energy of bilayer graphyne and to determine its most stable stacking mode. Our results show that inter-layer bindings of sp- and sp2-bonded carbon networks are significantly underestimated in a Kohn-Sham DFT approach, even with an exchange-correlation potential corrected to include, in some approximation, van der Waals interactions. Finally, our quantum Monte Carlo calculations reveal that the interlayer binding energy difference between the two stacking modes is only 0.9(4) eV/atom. From this we conclude that the two stable stacking modes of bilayer α-graphyne are almost degenerate with each other, and both will occur with about the same probability at room temperature unless there is a synthesis path that prefers one stacking over the other.« less

Carboxylic acid derivatives display potential selectivity for human histone deacetylase 6: Structure-based virtual screening, molecular docking and dynamics simulation studies.

PubMed

Uba, Abdullahi Ibrahim; Yelekçi, Kemal

2018-08-01

Human histone deacetylase 6 (HDAC6) has been shown to play a major role in oncogenic cell transformation via deacetylation of α-tubulin, making it a viable target of anticancer drug design and development. The crystal structure of HDAC6 catalytic domain 2 has been recently made available, providing avenues for structure-based drug design campaign. Here, in our continuous effort to identify potentially selective HDAC6 inhibitors, structure-based virtual screening of ∼72 461 compounds was carried out using Autodock Vina. The top 100 compounds with calculated ΔG < -10 kcal/mol were manually inspected for binding mode orientation. Furthermore, the top 20 compounds with reasonable binding modes were evaluated for selectivity by further docking against HDAC6 and HDAC7 using Autodock4. Four compounds with a carboxylic fragment, displayed potential selectivity for HDAC6 over HDAC7, and were found to have good druglike and ADMET properties. Their docking complexes were then submitted to 10 ns-molecular dynamics (MD) simulation using nanoscale MD (NAMD) software, to examine the stability of ligand binding modes. These predicted inhibitors remained bound to HDAC6 in the presence of water and ions, and the root-mean-square deviation (RMSD), radius of gyration (Rg) and nonbond distance (protein-ligand) profiles suggested that they might be stable over time of the simulation. This study may provide scaffolds for further lead optimization towards the design of HDAC6 inhibitors with improved selectivity. Copyright © 2018 Elsevier Ltd. All rights reserved.

The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange

PubMed Central

Qian, Yufeng; Johnson, Kenneth A.

2017-01-01

The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB–ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB)30 and (SSB)60, defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB)60 and (SSB)30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 109 m−1 s−1) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. PMID:28615444

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72.

PubMed

Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B

2018-01-22

An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

Structure of the Trehalose-6-phosphate Phosphatase from Brugia malayi Reveals Key Design Principles for Anthelmintic Drugs

PubMed Central

Farelli, Jeremiah D.; Galvin, Brendan D.; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E.; Causey, Thomas B.; Ali-Reynolds, Alana; Saltzberg, Daniel J.; Carlow, Clotilde K. S.; Dunaway-Mariano, Debra; Allen, Karen N.

2014-01-01

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible. PMID:24992307

The Mode of Inhibitor Binding to Peptidyl-tRNA Hydrolase: Binding Studies and Structure Determination of Unbound and Bound Peptidyl-tRNA Hydrolase from Acinetobacter baumannii

PubMed Central

Kaushik, Sanket; Singh, Nagendra; Yamini, Shavait; Singh, Avinash; Sinha, Mau; Arora, Ashish; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

2013-01-01

The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design. PMID:23844024

Structural Basis of Resistance to Anti-Cytochrome bc1 Complex Inhibitors: Implication for Drug Improvement

PubMed Central

Esser, Lothar; Yu, Chang-An; Xia, Di

2016-01-01

The emergence of drug resistance has devastating economic and social consequences, a testimonial of which is the rise and fall of inhibitors against the respiratory component cytochrome bc1 complex, a time tested and highly effective target for disease control. Unfortunately, the mechanism of resistance is a multivariate problem, including primarily mutations in the gene of the cytochrome b subunit but also activation of alternative pathways of ubiquinol oxidation and pharmacokinetic effects. There is a considerable interest in designing new bc1 inhibitors with novel modes of binding and lower propensity to induce the development of resistance. The accumulation of crystallographic data of bc1 complexes with and without inhibitors bound provides the structural basis for rational drug design. In particular, the cytochrome b subunit offers two distinct active sites that can be targeted for inhibition - the quinol oxidation site and the quinone reduction site. This review brings together available structural information of inhibited bc1 by various quinol oxidation- and reduction-site inhibitors, the inhibitor binding modes, conformational changes upon inhibitor binding of side chains in the active site and large scale domain movements of the iron-sulfur protein subunit. Structural data analysis provides a clear understanding of where and why existing inhibitors fail and points towards promising alternatives. PMID:23688079

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

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

Albright, Seth; Chen Bin; Holbrook, Kristen

CD14 functions as a key pattern recognition receptor for a diverse array of Gram-negative and Gram-positive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy, the binding of three different endotoxin ligands, lipopolysaccharide, lipoteichoic acid, and a PGN-derived compound, muramyl dipeptide to a {sup 15}N isotopically labeled 152-residue N-terminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern ofmore » residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site.« less

Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease.

PubMed

Li, M; Morris, G M; Lee, T; Laco, G S; Wong, C H; Olson, A J; Elder, J H; Wlodawer, A; Gustchina, A

2000-01-01

Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.

G Protein-Coupled Estrogen Receptor (GPER) Agonist Dual Binding Mode Analyses toward Understanding of its Activation Mechanism: A Comparative Homology Modeling Approach.

PubMed

Arnatt, Christopher K; Zhang, Yan

2013-07-01

G protein-coupled estrogen receptor (GPER) has been shown to be important in several disease states such as estrogen sensitive cancers. While several selective ligands have been identified for the receptor, little is known about how they interact with GPER and how their structures influence their activity. Specifically, within one series of ligands, whose structure varied only at one position, the replacement of a hydrogen atom with an acetyl group changed a potent antagonist into a potent agonist. In this study, two GPER homology models were constructed based on the x-ray crystal structures of both the active and inactive β 2 -adrenergic receptors (β 2 AR) in an effort to characterize the differences of binding modes between agonists and antagonists to the receptor, and to understand their activity in relation to their structures. The knowledge attained in this study is expected to provide valuable information on GPER ligands structure activity relationship to benefit future rational design of potent agonists and antagonists of the receptor for potential therapeutic applications.

G Protein-Coupled Estrogen Receptor (GPER) Agonist Dual Binding Mode Analyses toward Understanding of its Activation Mechanism: A Comparative Homology Modeling Approach

PubMed Central

Arnatt, Christopher K.; Zhang, Yan

2015-01-01

G protein-coupled estrogen receptor (GPER) has been shown to be important in several disease states such as estrogen sensitive cancers. While several selective ligands have been identified for the receptor, little is known about how they interact with GPER and how their structures influence their activity. Specifically, within one series of ligands, whose structure varied only at one position, the replacement of a hydrogen atom with an acetyl group changed a potent antagonist into a potent agonist. In this study, two GPER homology models were constructed based on the x-ray crystal structures of both the active and inactive β2-adrenergic receptors (β2AR) in an effort to characterize the differences of binding modes between agonists and antagonists to the receptor, and to understand their activity in relation to their structures. The knowledge attained in this study is expected to provide valuable information on GPER ligands structure activity relationship to benefit future rational design of potent agonists and antagonists of the receptor for potential therapeutic applications. PMID:26229572

Characteristic vibration patterns of odor compounds from bread-baking volatiles upon protein binding: density functional and ONIOM study and principal component analysis.

PubMed

Treesuwan, Witcha; Hirao, Hajime; Morokuma, Keiji; Hannongbua, Supa

2012-05-01

As the mechanism underlying the sense of smell is unclear, different models have been used to rationalize structure-odor relationships. To gain insight into odorant molecules from bread baking, binding energies and vibration spectra in the gas phase and in the protein environment [7-transmembrane helices (7TMHs) of rhodopsin] were calculated using density functional theory [B3LYP/6-311++G(d,p)] and ONIOM [B3LYP/6-311++G(d,p):PM3] methods. It was found that acetaldehyde ("acid" category) binds strongly in the large cavity inside the receptor, whereas 2-ethyl-3-methylpyrazine ("roasted") binds weakly. Lys296, Tyr268, Thr118 and Ala117 were identified as key residues in the binding site. More emphasis was placed on how vibrational frequencies are shifted and intensities modified in the receptor protein environment. Principal component analysis (PCA) suggested that the frequency shifts of C-C stretching, CH(3) umbrella, C = O stretching and CH(3) stretching modes have a significant effect on odor quality. In fact, the frequency shifts of the C-C stretching and C = O stretching modes, as well as CH(3) umbrella and CH(3) symmetric stretching modes, exhibit different behaviors in the PCA loadings plot. A large frequency shift in the CH(3) symmetric stretching mode is associated with the sweet-roasted odor category and separates this from the acid odor category. A large frequency shift of the C-C stretching mode describes the roasted and oily-popcorn odor categories, and separates these from the buttery and acid odor categories.

Pharmacophore-based virtual screening, biological evaluation and binding mode analysis of a novel protease-activated receptor 2 antagonist

NASA Astrophysics Data System (ADS)

Cho, Nam-Chul; Seo, Seoung-Hwan; Kim, Dohee; Shin, Ji-Sun; Ju, Jeongmin; Seong, Jihye; Seo, Seon Hee; Lee, Iiyoun; Lee, Kyung-Tae; Kim, Yun Kyung; No, Kyoung Tai; Pae, Ae Nim

2016-08-01

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR2 antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR2 antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22 µM against PAR2. In next step, a PAR2 homology model was constructed using the crystal structure of the PAR1 as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR2 agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe2435.39 is crucial for PAR2 ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR2 antagonist blocked a conformational rearrangement of Na+ allosteric site in contrast to PAR2 agonist that showed Na+ relocation upon GPCR activation. In conclusion, we identified new a PAR2 antagonist together with its binding mode, which provides useful insights for the design and development of PAR2 ligands.

Identifying the binding mode of a molecular scaffold

NASA Astrophysics Data System (ADS)

Chema, Doron; Eren, Doron; Yayon, Avner; Goldblum, Amiram; Zaliani, Andrea

2004-01-01

We describe a method for docking of a scaffold-based series and present its advantages over docking of individual ligands, for determining the binding mode of a molecular scaffold in a binding site. The method has been applied to eight different scaffolds of protein kinase inhibitors (PKI). A single analog of each of these eight scaffolds was previously crystallized with different protein kinases. We have used FlexX to dock a set of molecules that share the same scaffold, rather than docking a single molecule. The main mode of binding is determined by the mode of binding of the largest cluster among the docked molecules that share a scaffold. Clustering is based on our `nearest single neighbor' method [J. Chem. Inf. Comput. Sci., 43 (2003) 208-217]. Additional criteria are applied in those cases in which more than one significant binding mode is found. Using the proposed method, most of the crystallographic binding modes of these scaffolds were reconstructed. Alternative modes, that have not been detected yet by experiments, could also be identified. The method was applied to predict the binding mode of an additional molecular scaffold that was not yet reported and the predicted binding mode has been found to be very similar to experimental results for a closely related scaffold. We suggest that this approach be used as a virtual screening tool for scaffold-based design processes.

Structural basis of Zika virus helicase in recognizing its substrates.

PubMed

Tian, Hongliang; Ji, Xiaoyun; Yang, Xiaoyun; Zhang, Zhongxin; Lu, Zuokun; Yang, Kailin; Chen, Cheng; Zhao, Qi; Chi, Heng; Mu, Zhongyu; Xie, Wei; Wang, Zefang; Lou, Huiqiang; Yang, Haitao; Rao, Zihe

2016-08-01

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn(2+) and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn(2+). The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.

Structural basis for diversity in the SAM clan of riboswitches.

PubMed

Trausch, Jeremiah J; Xu, Zhenjiang; Edwards, Andrea L; Reyes, Francis E; Ross, Phillip E; Knight, Rob; Batey, Robert T

2014-05-06

In bacteria, sulfur metabolism is regulated in part by seven known families of riboswitches that bind S-adenosyl-l-methionine (SAM). Direct binding of SAM to these mRNA regulatory elements governs a downstream secondary structural switch that communicates with the transcriptional and/or translational expression machinery. The most widely distributed SAM-binding riboswitches belong to the SAM clan, comprising three families that share a common SAM-binding core but differ radically in their peripheral architecture. Although the structure of the SAM-I member of this clan has been extensively studied, how the alternative peripheral architecture of the other families supports the common SAM-binding core remains unknown. We have therefore solved the X-ray structure of a member of the SAM-I/IV family containing the alternative "PK-2" subdomain shared with the SAM-IV family. This structure reveals that this subdomain forms extensive interactions with the helix housing the SAM-binding pocket, including a highly unusual mode of helix packing in which two helices pack in a perpendicular fashion. Biochemical and genetic analysis of this RNA reveals that SAM binding induces many of these interactions, including stabilization of a pseudoknot that is part of the regulatory switch. Despite strong structural similarity between the cores of SAM-I and SAM-I/IV members, a phylogenetic analysis of sequences does not indicate that they derive from a common ancestor.

Deciphering the groove binding modes of tau-fluvalinate and flumethrin with calf thymus DNA

NASA Astrophysics Data System (ADS)

Tao, Mo; Zhang, Guowen; Pan, Junhui; Xiong, Chunhong

2016-02-01

Tau-fluvalinate (TFL) and flumethrin (FL), widely used in agriculture and a class of synthetic pyrethroid pesticides with a similar structure, may cause a potential security risk. Herein, the modes of binding in vitro of TFL and FL with calf thymus DNA (ctDNA) were characterized by fluorescence, UV-vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy with the aid of viscosity measurements, melting analyses and molecular docking studies. The fluorescence titration indicated that both TFL and FL bound to ctDNA forming complexes through hydrogen bonding and van der Waals forces. The binding constants of TFL and FL with ctDNA were in the range of 104 L mol- 1, and FL exhibited a higher binding propensity than TFL. The iodide quenching effect, single/double-stranded DNA effects, and ctDNA melting and viscosity measurements demonstrated that the binding of both TFL and FL to ctDNA was groove mode. The FT-IR analyses suggested the A-T region of the minor groove of ctDNA as the preferential binding for TFL and FL, which was confirmed by the displacement assays with Hoechst 33258 probe, and the molecular docking visualized the specific binding. The changes in CD spectra indicated that both FL and TFL induced the perturbation on the base stacking and helicity of B-DNA, but the disturbance caused by FL was more obvious. Gel electrophoresis analyses indicated that both TFL and FL did not cause significant DNA cleavage. This study provides novel insights into the binding properties of TFL/FL with ctDNA and its toxic mechanisms.

Nucleic Acid Database (NDB)

Science.gov Websites

the NDB archive or in the Non-Redundant list Advanced Search Search for structures based on structural features, chemical features, binding modes, citation and experimental information Featured Tools RNA 3D Motif Atlas, a representative collection of RNA 3D internal and hairpin loop motifs Non-redundant Lists

Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA-Encoded Chemical Library.

PubMed

Cuozzo, John W; Centrella, Paolo A; Gikunju, Diana; Habeshian, Sevan; Hupp, Christopher D; Keefe, Anthony D; Sigel, Eric A; Soutter, Holly H; Thomson, Heather A; Zhang, Ying; Clark, Matthew A

2017-05-04

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding.

PubMed

Cash, Jennifer N; Angerman, Elizabeth B; Kattamuri, Chandramohan; Nolan, Kristof; Zhao, Huaying; Sidis, Yisrael; Keutmann, Henry T; Thompson, Thomas B

2012-01-06

TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

NASA Astrophysics Data System (ADS)

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-03-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses.

Structural insights into Cydia pomonella pheromone binding protein 2 mediated prediction of potentially active semiochemicals

PubMed Central

Tian, Zhen; Liu, Jiyuan; Zhang, Yalin

2016-01-01

Given the advantages of behavioral disruption application in pest control and the damage of Cydia pomonella, due progresses have not been made in searching active semiochemicals for codling moth. In this research, 31 candidate semiochemicals were ranked for their binding potential to Cydia pomonella pheromone binding protein 2 (CpomPBP2) by simulated docking, and this sorted result was confirmed by competitive binding assay. This high predicting accuracy of virtual screening led to the construction of a rapid and viable method for semiochemicals searching. By reference to binding mode analyses, hydrogen bond and hydrophobic interaction were suggested to be two key factors in determining ligand affinity, so is the length of molecule chain. So it is concluded that semiochemicals of appropriate chain length with hydroxyl group or carbonyl group at one head tended to be favored by CpomPBP2. Residues involved in binding with each ligand were pointed out as well, which were verified by computational alanine scanning mutagenesis. Progress made in the present study helps establish an efficient method for predicting potentially active compounds and prepares for the application of high-throughput virtual screening in searching semiochemicals by taking insights into binding mode analyses. PMID:26928635

A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes

PubMed Central

Yu, Xinchao; Breitman, Marianna; Goldberg, Jonathan

2012-01-01

Summary Budding of COPI-coated vesicles from Golgi membranes requires an Arf-family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the γ- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P2-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules. PMID:22304919

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

Conformational changes in the expression domain of the Escherichia coli thiM riboswitch

PubMed Central

Rentmeister, Andrea; Mayer, Günter; Kuhn, Nicole; Famulok, Michael

2007-01-01

The thiM riboswitch contains an aptamer domain that adaptively binds the coenzyme thiamine pyrophosphate (TPP). The binding of TPP to the aptamer domain induces structural rearrangements that are relayed to a second domain, the so-called expression domain, thereby interfering with gene expression. The recently solved crystal structures of the aptamer domains of the thiM riboswitches in complex with TPP revealed how TPP stabilizes secondary and tertiary structures in the RNA ligand complex. To understand the global modes of reorganization between the two domains upon metabolite binding the structure of the entire riboswitch in presence and absence of TPP needs to be determined. Here we report the secondary structure of the entire thiM riboswitch from Escherichia coli in its TPP-free form and its transition into the TPP-bound variant, thereby depicting domains of the riboswitch that serve as communication links between the aptamer and the expression domain. Furthermore, structural probing provides an explanation for the lack of genetic control exerted by a riboswitch variant with mutations in the expression domain that still binds TPP. PMID:17517779

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Docking and Hydropathic Scoring of Polysubstituted Pyrrole Compounds with Anti-Tubulin Activity

PubMed Central

Tripathi, Ashutosh; Fornabaio, Micaela; Kellogg, Glen E.; Gupton, John T.; Gewirtz, David A.; Yeudall, W. Andrew; Vega, Nina E.; Mooberry, Susan L.

2008-01-01

Compounds that bind at the colchicine site of tubulin have drawn considerable attention with studies indicating that these agents suppress microtubule dynamics and inhibit tubulin polymerization. Data for eighteen polysubstituted pyrrole compounds are reported, including antiproliferative activity against human MDA-MB-435 cells and calculated free energies of binding following docking the compounds into models of αβ-tubulin. These docking calculations coupled with HINT interaction analyses are able to represent the complex structures and the binding modes of inhibitors such that calculated and measured free energies of binding correlate with an r2 of 0.76. Structural analysis of the binding pocket identifies important intermolecular contacts that mediate binding. As seen experimentally, the complex with JG-03-14 (3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2- carboxylic acid ethyl ester) is the most stable. These results illuminate the binding process and should be valuable in the design of new pyrrole-based colchicine site inhibitors as these compounds have very accessible syntheses. PMID:18083520

Structure of D-AKAP2:PKA RI complex: Insights into AKAP specificity and selectivity

PubMed Central

Sarma, Ganapathy N.; Kinderman, Francis S.; Kim, Choel; von Daake, Sventja; Chen, Lirong; Wang, Bi-Cheng; Taylor, Susan S.

2011-01-01

Summary A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here, we have determined the structures of the RIα D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-termina helix and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RIIα:D-AKAP2 complex structure makes the mode of binding to RIα novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity. PMID:20159461

Structural analysis of the binding modes of minor groove ligands comprised of disubstituted benzenes

PubMed Central

Hawkins, Cheryl A.; Watson, Charles; Yan, Yinfa; Gong, Bing; Wemmer, David E.

2001-01-01

Two-dimensional homonuclear NMR was used to characterize synthetic DNA minor groove-binding ligands in complexes with oligonucleotides containing three different A-T binding sites. The three ligands studied have a C2 axis of symmetry and have the same general structural motif of a central para-substituted benzene ring flanked by two meta-substituted rings, giving the molecules a crescent shape. As with other ligands of this shape, specificity seems to arise from a tight fit in the narrow minor groove of the preferred A-T-rich sequences. We found that these ligands slide between binding subsites, behavior attributed to the fact that all of the amide protons in the ligand backbone cannot hydrogen bond to the minor groove simultaneously. PMID:11160926

A new cationic porphyrin derivative (TMPipEOPP) with large side arm substituents: a highly selective G-quadruplex optical probe.

PubMed

Zhu, Li-Na; Zhao, Shu-Juan; Wu, Bin; Li, Xiao-Zeng; Kong, De-Ming

2012-01-01

The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA) sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4), interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1-piperidinyl)ethoxy]phenyl} porphyrin (TMPipEOPP), with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.

T-Cell Receptors Binding Orientation over Peptide/MHC Class I Is Driven by Long-Range Interactions

PubMed Central

Ferber, Mathias; Zoete, Vincent; Michielin, Olivier

2012-01-01

Crystallographic data about T-Cell Receptor – peptide – major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes. PMID:23251658

T-cell receptors binding orientation over peptide/MHC class I is driven by long-range interactions.

PubMed

Ferber, Mathias; Zoete, Vincent; Michielin, Olivier

2012-01-01

Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

The structure of transcription termination factor Nrd1 reveals an original mode for GUAA recognition

PubMed Central

Franco-Echevarría, Elsa; González-Polo, Noelia; Zorrilla, Silvia; Martínez-Lumbreras, Santiago; Santiveri, Clara M.; Campos-Olivas, Ramón; Sánchez, Mar; Calvo, Olga

2017-01-01

Abstract Transcription termination of non-coding RNAs is regulated in yeast by a complex of three RNA binding proteins: Nrd1, Nab3 and Sen1. Nrd1 is central in this process by interacting with Rbp1 of RNA polymerase II, Trf4 of TRAMP and GUAA/G terminator sequences. We lack structural data for the last of these binding events. We determined the structures of Nrd1 RNA binding domain and its complexes with three GUAA-containing RNAs, characterized RNA binding energetics and tested rationally designed mutants in vivo. The Nrd1 structure shows an RRM domain fused with a second α/β domain that we name split domain (SD), because it is formed by two non-consecutive segments at each side of the RRM. The GUAA interacts with both domains and with a pocket of water molecules, trapped between the two stacking adenines and the SD. Comprehensive binding studies demonstrate for the first time that Nrd1 has a slight preference for GUAA over GUAG and genetic and functional studies suggest that Nrd1 RNA binding domain might play further roles in non-coding RNAs transcription termination. PMID:28973465

Structure-Based Design, Synthesis, Evaluation And Crystal Structures of Transition State Analogue Inhibitors of Inosine Monophosphate Cyclohydrolase

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

Xu, L.; Chong, Y.; Hwang, I.

2007-07-13

The inosine monophosphate cyclohydrolase (IMPCH) component (residues 1-199) of the bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase, residues 200-593)/IMPCH (ATIC) catalyzes the final step in the de novo purine biosynthesis pathway that produces IMP. As a potential target for antineoplastic intervention, we designed IMPCH inhibitors, 1,5-dihydroimidazo[4,5-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (heterocycle, 1), the corresponding nucleoside (2), and the nucleoside monophosphate (nucleotide) (3), as mimics of the tetrahedral intermediate in the cyclization reaction. All compounds are competitive inhibitors against IMPCH (K(i) values = 0.13-0.23 microm) with the simple heterocycle 1 exhibiting the most potent inhibition (K(i) = 0.13 microm). Crystal structures of bifunctional ATICmore » in complex with nucleoside 2 and nucleotide 3 revealed IMPCH binding modes similar to that of the IMPCH feedback inhibitor, xanthosine 5'-monophosphate. Surprisingly, the simpler heterocycle 1 had a completely different IMPCH binding mode and was relocated to the phosphate binding pocket that was identified from previous xanthosine 5'-monophosphate structures. The aromatic imidazole ring interacts with a helix dipole, similar to the interaction with the phosphate moiety of 3. The crystal structures not only revealed the mechanism of inhibition of these compounds, but they now serve as a platform for future inhibitor improvements. Importantly, the nucleoside-complexed structure supports the notion that inhibitors lacking a negatively charged phosphate can still inhibit IMPCH activity with comparable potency to phosphate-containing inhibitors. Provocatively, the nucleotide inhibitor 3 also binds to the AICAR Tfase domain of ATIC, which now provides a lead compound for the design of inhibitors that simultaneously target both active sites of this bifunctional enzyme.« less

Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

PubMed

Yang, Zhenlin; Han, Shuo; Keller, Max; Kaiser, Anette; Bender, Brian J; Bosse, Mathias; Burkert, Kerstin; Kögler, Lisa M; Wifling, David; Bernhardt, Guenther; Plank, Nicole; Littmann, Timo; Schmidt, Peter; Yi, Cuiying; Li, Beibei; Ye, Sheng; Zhang, Rongguang; Xu, Bo; Larhammar, Dan; Stevens, Raymond C; Huster, Daniel; Meiler, Jens; Zhao, Qiang; Beck-Sickinger, Annette G; Buschauer, Armin; Wu, Beili

2018-04-01

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y 1 , Y 2 , Y 4 and Y 5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y 1 receptor (Y 1 R) 4 . A number of peptides and small-molecule compounds have been characterized as Y 1 R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y 1 R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y 1 R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y 1 R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y 1 R can enable structure-based drug discovery that targets NPY receptors.

Change of the binding mode of the DNA/proflavine system induced by ethanol.

PubMed

García, Begoña; Leal, José M; Ruiz, Rebeca; Biver, Tarita; Secco, Fernando; Venturini, M

2010-07-01

The equilibria and kinetics of the binding of proflavine to poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) were investigated in ethanol/water mixtures using spectrophotometric, circular dichroism, viscometric, and T-jump methods. All methods concur in showing that two modes of interaction are operative: intercalation and surface binding. The latter mode is favored by increasing ethanol and/or the proflavine content. Both static and kinetic experiments show that, concerning the poly(dG-dC).poly(dG-dC)/proflavine system, intercalation largely prevails up to 20% EtOH. For higher EtOH levels surface binding becomes dominant. Concerning the poly(dA-dT).poly(dA-dT)/proflavine system, melting experiments show that addition of proflavine stabilizes the double stranded structure, but the effect is reduced in the presence of EtOH. The DeltaH degrees and DeltaS degrees values of the melting process, measured at different concentrations of added proflavine, are linearly correlated, revealing the presence of the enthalpy-entropy compensation phenomenon (EEC). The nonmonotonicity of the "entropic term" of the EEC reveals the transition between the two binding modes. T-jump experiments show two relaxation effects, but at the highest levels of EtOH (>25%) the kinetic curves become monophasic, confirming the prevalence of the surface complex. A branched mechanism is proposed where diffusion controlled formation of a precursor complex occurs in the early stage of the binding process. This evolves toward the surface and/or the intercalated complex according to two rate-determining parallel steps. CD spectra suggest that, in the surface complex, proflavine is bound to DNA in the form of an aggregate.

Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast*

PubMed Central

Lee, Jun Hyuck; Park, Ae Kyung; Do, Hackwon; Park, Kyoung Sun; Moh, Sang Hyun; Chi, Young Min; Kim, Hak Jun

2012-01-01

Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of ∼25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57- and 2.43-Å resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96–115 form a long α-helix that packs along one face of the β-helix), and a C-terminal hydrophobic loop region (243PFVPAPEVV251). Unexpectedly, the C-terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical hyperactive antifreeze proteins, the ice-binding site is more complex and does not have a simple ice-binding motif. In conclusion, we could identify the ice-binding site of LeIBP and discuss differences in the ice-binding modes compared with other known antifreeze proteins and ice-binding proteins. PMID:22303017

Flexibility and adaptability in binding of E. coli cytidine repressor to different operators suggests a role in differential gene regulation.

PubMed

Tretyachenko-Ladokhina, Vira; Cocco, Melanie J; Senear, Donald F

2006-09-15

Interactions between DNA-bound transcription factors CytR and CRP regulate the promoters of the Escherichia coli CytR regulon. A distinctive feature of the palindromic CytR operators is highly variable length central spacers (0-9 bp). Previously we demonstrated distinct modes of CytR binding to operators that differ in spacer length. These different modes are characterized by opposite enthalpic and entropic contributions at 25 degrees C. Of particular note were radically different negative DeltaCp values suggesting variable contribution from coupled protein folding and/or DNA structural transitions. We proposed that the CytR DNA binding-domain adopts either a more rigid or flexible DNA-bound conformation in response to the different spacer lengths. More recently, similar effects were shown to contribute to discrimination between operator and non-specific DNA binding by LacR, a CytR homolog. Here we have extended the thermodynamic analysis to the remaining natural CytR operators plus a set of synthetic operators designed to isolate spacing as the single variable. The thermodynamic results show a broad and monotonic range of effects that are primarily dependent on spacer length. The magnitude of effects suggests participation by more than the DNA-binding domain. 15N HSQC NMR and CD spectral analyses were employed to characterize the structural basis for these effects. The results indicate that while CytR forms a well-ordered structure in solution, it is highly dynamic. We propose a model in which a large ensemble of native state conformations narrows upon binding, to an extent governed by operator spacing. This in turn is expected to constrain intermolecular interactions in the CytR-CRP-DNA complex, thus generating operator-specific effects on repression and induction of transcription.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

PubMed

Bohl, Casey E; Wu, Zengru; Chen, Jiyun; Mohler, Michael L; Yang, Jun; Hwang, Dong Jin; Mustafa, Suni; Miller, Duane D; Bell, Charles E; Dalton, James T

2008-10-15

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Key Structures and Interactions for Binding of Mycobacterium tuberculosis Protein Kinase B Inhibitors from Molecular Dynamics Simulation.

PubMed

Punkvang, Auradee; Kamsri, Pharit; Saparpakorn, Patchreenart; Hannongbua, Supa; Wolschann, Peter; Irle, Stephan; Pungpo, Pornpan

2015-07-01

Substituted aminopyrimidine inhibitors have recently been introduced as antituberculosis agents. These inhibitors show impressive activity against protein kinase B, a Ser/Thr protein kinase that is essential for cell growth of M. tuberculosis. However, up to now, X-ray structures of the protein kinase B enzyme complexes with the substituted aminopyrimidine inhibitors are currently unavailable. Consequently, structural details of their binding modes are questionable, prohibiting the structural-based design of more potent protein kinase B inhibitors in the future. Here, molecular dynamics simulations, in conjunction with molecular mechanics/Poisson-Boltzmann surface area binding free-energy analysis, were employed to gain insight into the complex structures of the protein kinase B inhibitors and their binding energetics. The complex structures obtained by the molecular dynamics simulations show binding free energies in good agreement with experiment. The detailed analysis of molecular dynamics results shows that Glu93, Val95, and Leu17 are key residues responsible to the binding of the protein kinase B inhibitors. The aminopyrazole group and the pyrimidine core are the crucial moieties of substituted aminopyrimidine inhibitors for interaction with the key residues. Our results provide a structural concept that can be used as a guide for the future design of protein kinase B inhibitors with highly increased antagonistic activity. © 2014 John Wiley & Sons A/S.

Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions

PubMed Central

Joseph, Prem Raj B.; Mosier, Philip D.; Desai, Umesh R.; Rajarathnam, Krishna

2015-01-01

Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8–GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer–GAG interactions and function. PMID:26371375

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

PubMed

Seufert, Florian; Kuhn, Maximilian; Hein, Michael; Weiwad, Matthias; Vivoli, Mirella; Norville, Isobel H; Sarkar-Tyson, Mitali; Marshall, Laura E; Schweimer, Kristian; Bruhn, Heike; Rösch, Paul; Harmer, Nicholas J; Sotriffer, Christoph A; Holzgrabe, Ulrike

2016-11-01

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis, crystal structure analysis, molecular docking studies and density functional theory predictions of the local reactive properties and degradation properties of a novel halochalcone

NASA Astrophysics Data System (ADS)

Arshad, Suhana; Pillai, Renjith Raveendran; Zainuri, Dian Alwani; Khalib, Nuridayanti Che; Razak, Ibrahim Abdul; Armaković, Stevan; Armaković, Sanja J.

2017-09-01

In the present study, single crystals of E)-3-(3,5-dichlorophenyl)-1-(4-fluorophenyl)prop-2-en-1-one, were prepared and structurally characterized by single crystal X-ray diffraction analysis. The molecular structure crystallized in monoclinic crystal system with P21/c space group. Sensitivity of the title molecule towards electrophilic attacks has been examined by calculations of average localized ionization energies (ALIE) and their mapping to electron density surface. Further determination of atoms that could be important reactive centres has been performed by calculations of Fukui functions. Sensitivity of title molecule towards autoxidation and hydrolysis mechanisms has been assessed by calculations of bond dissociation energies and radial distribution functions (RDF), respectively. Also, in order to explore possible binding mode of the title compound towards Dihydrofolate reductase enzyme, we have utilized in silico molecular docking to explore possible binding modes of the title compound with the DHFR enzyme.

Unveiling the molecular mechanism of brassinosteroids: Insights from structure-based molecular modeling studies.

PubMed

Lei, Beilei; Liu, Jiyuan; Yao, Xiaojun

2015-12-01

Brassinosteroid (BR) phytohormones play indispensable roles in plant growth and development. Brassinolide (BL) and 24-epibrassinolide (24-epiBL) are the most active ones among the BRs reported thus far. Unfortunately, the extremely low natural content and intricate synthesis process limit their popularization in agricultural production. Earlier reports to discover alternative compounds have resulted in molecules with nearly same scaffold structure and without diversity in chemical space. In the present study, receptors structure based BRs regulation mechanism was analyzed. First, we examined the detailed binding interactions and their dynamic stability between BL and its receptor BRI1 and co-receptor BAK1. Then, the binding modes and binding free energies for 24-epiBL and a series of representative BRs binding with BRI1 and BRI1-BAK1 were carried out by molecular docking, energy minimization and MM-PBSA free energy calculation. The obtained binding structures and energetic results provided vital insights into the structural factors affecting the activity from both receptors and BRs aspects. Subsequently, the obtained knowledge will serve as valuable guidance to build pharmacophore models for rational screening of new scaffold alternative BRs. Copyright © 2015 Elsevier Inc. All rights reserved.

Structural insights into binding of small molecule inhibitors to Enhancer of Zeste Homolog 2

NASA Astrophysics Data System (ADS)

Kalinić, Marko; Zloh, Mire; Erić, Slavica

2014-11-01

Enhancer of Zeste Homolog 2 (EZH2) is a SET domain protein lysine methyltransferase (PKMT) which has recently emerged as a chemically tractable and therapeutically promising epigenetic target, evidenced by the discovery and characterization of potent and highly selective EZH2 inhibitors. However, no experimental structures of the inhibitors co-crystallized to EZH2 have been resolved, and the structural basis for their activity and selectivity remains unknown. Considering the need to minimize cross-reactivity between prospective PKMT inhibitors, much can be learned from understanding the molecular basis for selective inhibition of EZH2. Thus, to elucidate the binding of small-molecule inhibitors to EZH2, we have developed a model of its fully-formed cofactor binding site and used it to carry out molecular dynamics simulations of protein-ligand complexes, followed by molecular mechanics/generalized born surface area calculations. The obtained results are in good agreement with biochemical inhibition data and reflect the structure-activity relationships of known ligands. Our findings suggest that the variable and flexible post-SET domain plays an important role in inhibitor binding, allowing possibly distinct binding modes of inhibitors with only small variations in their structure. Insights from this study present a good basis for design of novel and optimization of existing compounds targeting the cofactor binding site of EZH2.

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.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

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

Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination ofmore » an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.« less

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

Nuclear Resonance Vibrational Spectroscopic Definition of Peroxy Intermediates in Nonheme Iron Sites

DOE PAGES

Sutherlin, Kyle D.; Liu, Lei V.; Lee, Yong-Min; ...

2016-11-02

Fe III-(hydro)peroxy intermediates have been isolated in two classes of mononuclear nonheme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well-defined, due to a lack of vibrational structural data. Nuclear resonance vibrational spectroscopy (NRVS) is an important technique for obtaining vibrational information on these and other intermediates, as it is sensitive to all normal modes with Fe displacement. Here in this paper, we present the NRVS spectra of side-on Fe III-peroxy and end-on Fe III-hydroperoxy model complexes and assignmore » these spectra using calibrated DFT calculations. We then use DFT calculations to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe–O–O angle. This study identifies four spectroscopic handles that will enable definition of the binding mode and protonation state of Fe III-peroxy intermediates in mononuclear nonheme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity.« less

Novel propanamides as fatty acid amide hydrolase inhibitors.

PubMed

Deplano, Alessandro; Morgillo, Carmine Marco; Demurtas, Monica; Björklund, Emmelie; Cipriano, Mariateresa; Svensson, Mona; Hashemian, Sanaz; Smaldone, Giovanni; Pedone, Emilia; Luque, F Javier; Cabiddu, Maria G; Novellino, Ettore; Fowler, Christopher J; Catalanotti, Bruno; Onnis, Valentina

2017-08-18

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Nuclear Resonance Vibrational Spectroscopic Definition of Peroxy Intermediates in Nonheme Iron Sites

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

Sutherlin, Kyle D.; Liu, Lei V.; Lee, Yong-Min

Fe III-(hydro)peroxy intermediates have been isolated in two classes of mononuclear nonheme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well-defined, due to a lack of vibrational structural data. Nuclear resonance vibrational spectroscopy (NRVS) is an important technique for obtaining vibrational information on these and other intermediates, as it is sensitive to all normal modes with Fe displacement. Here in this paper, we present the NRVS spectra of side-on Fe III-peroxy and end-on Fe III-hydroperoxy model complexes and assignmore » these spectra using calibrated DFT calculations. We then use DFT calculations to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe–O–O angle. This study identifies four spectroscopic handles that will enable definition of the binding mode and protonation state of Fe III-peroxy intermediates in mononuclear nonheme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity.« less

Nuclear Resonance Vibrational Spectroscopic Definition of Peroxy Intermediates in Nonheme Iron Sites.

PubMed

Sutherlin, Kyle D; Liu, Lei V; Lee, Yong-Min; Kwak, Yeonju; Yoda, Yoshitaka; Saito, Makina; Kurokuzu, Masayuki; Kobayashi, Yasuhiro; Seto, Makoto; Que, Lawrence; Nam, Wonwoo; Solomon, Edward I

2016-11-02

Fe III -(hydro)peroxy intermediates have been isolated in two classes of mononuclear nonheme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well-defined, due to a lack of vibrational structural data. Nuclear resonance vibrational spectroscopy (NRVS) is an important technique for obtaining vibrational information on these and other intermediates, as it is sensitive to all normal modes with Fe displacement. Here, we present the NRVS spectra of side-on Fe III -peroxy and end-on Fe III -hydroperoxy model complexes and assign these spectra using calibrated DFT calculations. We then use DFT calculations to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe-O-O angle. This study identifies four spectroscopic handles that will enable definition of the binding mode and protonation state of Fe III -peroxy intermediates in mononuclear nonheme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity.

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

PubMed

Das, Pratyusa; Chaudhari, Sunil Kumar; Das, Asmita; Kundu, Somashree; Saha, Chabita

2018-04-24

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 10 7  M -1 and 4.2 × 10 6  M -1 , respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

Spectroscopic and molecular modeling studies on the binding of the flavonoid luteolin and human serum albumin.

PubMed

Jurasekova, Zuzana; Marconi, Giancarlo; Sanchez-Cortes, Santiago; Torreggiani, Armida

2009-11-01

Luteolin (LUT) is a polyphenolic compound, found in a variety of fruits, vegetables, and seeds, which has a variety of pharmacological properties. In the present contribution, binding of LUT to human serum albumin (HSA), the most abundant carrier protein in the blood, was investigated with the aim of describing the binding mode and parameters of the interaction. The application of circular dichroism, UV-Vis absorption, fluorescence, Raman and surface-enhanced Raman scattering spectroscopy combined with molecular modeling afforded a clear picture of the association mode of LUT to HSA. Specific interactions with protein amino acids were evidenced. LUT was found to be associated in subdomain IIA where an interaction with Trp-214 is established. Hydrophobic and electrostatic interactions are the major acting forces in the binding of LUT to HSA. The HSA conformations were slightly altered by the drug complexation with reduction of alpha-helix and increase of beta-turns structures, suggesting a partial protein unfolding. Also the configuration of at least two disulfide bridges were altered. Furthermore, the study of molecular modeling afforded the binding geometry. 2009 Wiley Periodicals, Inc.

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

PubMed Central

Zhang, Zheng; Jakkaraju, Sriram; Blain, Joy; Gogol, Kenneth; Zhao, Lei; Hartley, Robert C.; Karlsson, Courtney A.; Staker, Bart L.; Stewart, Lance J.; Myler, Peter J.; Clare, Michael; Begley, Darren W.; Horn, James R.; Hagen, Timothy J

2013-01-01

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series. PMID:24157367

Study on control of defect mode in hybrid mirror chirped porous silicon photonic crystal

NASA Astrophysics Data System (ADS)

Chen, Ying; Luo, Pei; Han, Yangyang; Cui, Xingning; He, Lei

2018-03-01

Based on the optical resonance principle and the tight-binding theory, a hybrid mirror chirped porous silicon photonic crystal is proposed. The control of the defect mode in hybrid mirror chirped porous silicon photonic crystal is studied. Through the numerical simulation, the control regulations of the defect modes resulted by the number of the periodical layers for the fundamental unit and the cascading number of the chirped structures are analyzed, and the split and the degeneration of the defect modes resulted by the change of the relative location between the mirror structures and the quasi-mirror structures are discussed. The simulation results show that the band gap would be broadened with the increase of the chirp quantity and the layer number of unilateral chirp. Adjusting the structural parameters of the hybrid mirror structure, the multimode characteristics will occur in the band gap. The more the cascading number of the chirped units, the more the number of the filtering channels will be. In addition, with the increase of the relative location between the mirror structures and the quasi-mirror structures, the degeneration of the defect modes will occur and can obtain high Q value. The structure can provide effective theoretical references for the design the multi-channel filters and high Q value sensors.

Molecular docking studies of selected tricyclic and quinone derivatives on trypanothione reductase of Leishmania infantum.

PubMed

Venkatesan, Santhosh Kannan; Shukla, Anil Kumar; Dubey, Vikash Kumar

2010-10-01

Visceral leishmaniasis, most lethal form of Leishmaniasis, is caused by Leishmania infantum in the Old world. Current therapeutics for the disease is associated with a risk of high toxicity and development of drug resistant strains. Thiol-redox metabolism involving trypanothione and trypanothione reductase, key for survival of Leishmania, is a validated target for rational drug design. Recently published structure of trypanothione reductase (TryR) from L. infantum, in oxidized and reduced form along with Sb(III), provides vital clues on active site of the enzyme. In continuation with our attempts to identify potent inhibitors of TryR, we have modeled binding modes of selected tricyclic compounds and quinone derivatives, using AutoDock4. Here, we report a unique binding mode for quinone derivatives and 9-aminoacridine derivatives, at the FAD binding domain. A conserved hydrogen bonding pattern was observed in all these compounds with residues Thr335, Lys60, His461. With the fact that these residues aid in the orientation of FAD towards the active site forming the core of the FAD binding domain, designing selective and potent compounds that could replace FAD in vivo during the synthesis of Trypanothione reductase can be deployed as an effective strategy in designing new drugs towards Leishmaniasis. We also report the binding of Phenothiazine and 9-aminoacridine derivatives at the Z site of the protein. The biological significance and possible mode of inhibition by quinone derivatives, which binds to FAD binding domain, along with other compounds are discussed. (c) 2010 Wiley Periodicals, Inc.

CW EPR parameters reveal cytochrome P450 ligand binding modes.

PubMed

Lockart, Molly M; Rodriguez, Carlo A; Atkins, William M; Bowman, Michael K

2018-06-01

Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-H 2 O-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-bridged complexes are remarkably common among a range of nitrogenous drugs or drug fragments that bind to CYP3A4 or CYP2C9. Principal component analysis reveals a distinct clustering of CW EPR spectral parameters for water-bridged complexes. CW EPR reveals heterogeneous mixtures of ligated states, including multiple directly-coordinated complexes and water-bridged complexes. These results suggest that water-bridged complexes are under-represented in CYP structural databases and can have energies similar to other ligation modes. The data indicates that water-bridged binding modes can be identified and distinguished from directly-coordinated binding by CW EPR. Copyright © 2018 Elsevier Inc. All rights reserved.

Suppression of gyrase-mediated resistance by C7 aryl fluoroquinolones

PubMed Central

Malik, Muhammad; Mustaev, Arkady; Schwanz, Heidi A.; Luan, Gan; Shah, Nirali; Oppegard, Lisa M.; de Souza, Ernane C.; Hiasa, Hiroshi; Zhao, Xilin; Kerns, Robert J.; Drlica, Karl

2016-01-01

Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies show that quinolone binding involves a water/metal-ion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA (gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding mode in which the other end of the quinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance. PMID:26984528

Binding Leverage as a Molecular Basis for Allosteric Regulation

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design. PMID:21935347

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

PubMed

Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

2015-09-11

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction.

PubMed

Pant, Kiran; Chereau, David; Hatch, Victoria; Dominguez, Roberto; Lehman, William

2006-06-16

Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.

AgI -Induced Switching of DNA Binding Modes via Formation of a Supramolecular Metallacycle.

PubMed

Basak, Shibaji; Léon, J Christian; Ferranco, Annaleizle; Sharma, Renu; Hebenbrock, Marian; Lough, Alan; Müller, Jens; Kraatz, Heinz-Bernhard

2018-03-12

The histidine derivative L1 of the DNA intercalator naphthalenediimide (NDI) forms a triangular Ag I complex (C2). The interactions of L1 and of C2 with DNA were studied by circular dichroism (CD) and UV/Vis spectroscopy and by viscosity studies. Different binding modes were observed for L1 and for C2, as the Ag I complex C2 is too large in size to act as an intercalator. If Ag I is added to the NDI molecule that is already intercalated into a duplex, higher order complexes are formed within the DNA duplex and cause disruptions in the helical duplex structure, which leads to a significant decrease in the characteristic CD features of B-DNA. Thus, via addition of a metal we show how a classic and well-known organic intercalator unit can be turned into a partial metallo insertor. We also show how electrochemical impedance spectroscopy (EIS) can be used to probe DNA binding modes on DNA films that are immobilized on gold surfaces. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Binding mode and potency of N-indolyloxopyridinyl-4-aminopropanyl-based inhibitors targeting Trypanosoma cruzi CYP51

DOE PAGES

Vieira, Debora F.; Choi, Jun Yong; Calvet, Claudia M.; ...

2014-11-13

Chagas disease is a chronic infection in humans caused by Trypanosoma cruzi and manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Limited therapeutic options to prevent and treat Chagas disease put 8 million people infected with T. cruzi worldwide at risk. CYP51, involved in the biosynthesis of the membrane sterol component in eukaryotes, is a promising drug target in T. cruzi. We report the structure–activity relationships (SAR) of an N-arylpiperazine series of N-indolyloxopyridinyl-4-aminopropanyl-based inhibitors designed to probe the impact of substituents in the terminal N-phenyl ring on binding mode, selectivity and potency. Depending on the substituents at C-4, two distinct ringmore » binding modes, buried and solvent-exposed, have been observed by X-ray structure analysis (resolution of 1.95–2.48 Å). Lastly, the 5-chloro-substituted analogs 9 and 10 with no substituent at C-4 demonstrated improved selectivity and potency, suppressing ≥99.8% parasitemia in mice when administered orally at 25 mg/kg, b.i.d., for 4 days.« less

A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism

PubMed Central

Phakthanakanok, Krongsakda; Ratanakhanokchai, Khanok; Kyu, Khin Lay; Sompornpisut, Pornthep; Watts, Aaron; Pinitglang, Surapong

2009-01-01

Background SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach. Results The one turn α-helix chain (residues 47–54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide. Conclusion MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide. PMID:19208150

Synthesis and structure of a heptanuclear nickel(II) complex uniquely exhibiting four distinct binding modes, two of which are novel, for a hydroxamate ligand.

PubMed

Gaynor, Declan; Starikova, Zoya A; Ostrovsky, Sergei; Haase, Wolfgang; Nolan, Kevin B

2002-03-07

The reaction of 2-(dimethylamino)phenylhydroxamic acid (2-dmAphaH) with NiSO(4).6H2O gives the complex [Ni7(2-dmAphaH-1)2(2-dmApha)8(H2O)2]SO(4).15H2O uniquely exhibiting four distinct hydroxamate binding modes, two of which are novel, and showing both antiferromagnetic and ferromagnetic interactions in contrast to [Cu5(2-dmAphaH-1)4(HSO4)2(MeOH)2].2MeOH, a strongly antiferromagnetic metallacrown formed with CuSO(4).5H2O.

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+•Calmodulin Displays a Unique Binding Mode*

PubMed Central

Liu, Yanshun; Zheng, Xunhai; Mueller, Geoffrey A.; Sobhany, Mack; DeRose, Eugene F.; Zhang, Yingpei; London, Robert E.; Birnbaumer, Lutz

2012-01-01

Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca2+-depletion sensor STIM1. This is followed by a fast Ca2+·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp76 of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1. PMID:23109337

Spectroscopic and molecular docking studies on the interaction of antiviral drug nevirapine with calf thymus DNA.

PubMed

Moghadam, Neda Hosseinpour; Salehzadeh, Sadegh; Shahabadi, Nahid

2017-09-02

The interaction of calf thymus DNA with nevirapine at physiological pH was studied by using absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, salt effect studies and computational methods. The drug binds to ct-DNA in a groove binding mode, as shown by slight variation in the viscosity of ct-DNA. Furthermore, competitive fluorimetric studies with Hoechst 33258 indicate that nevirapine binds to DNA via groove binding. Moreover, the structure of nevirapine was optimized by DFT calculations and was used for the molecular docking calculations. The molecular docking results suggested that nevirapine prefers to bind on the minor groove of ct-DNA.

Structural and Biochemical Analyses of Glycoside Hydrolase Families 5 and 26 β-(1,4)-Mannanases from Podospora anserina Reveal Differences upon Manno-oligosaccharide Catalysis*

PubMed Central

Couturier, Marie; Roussel, Alain; Rosengren, Anna; Leone, Philippe; Stålbrand, Henrik; Berrin, Jean-Guy

2013-01-01

The microbial deconstruction of the plant cell wall is a key biological process that is of increasing importance with the development of a sustainable biofuel industry. The glycoside hydrolase families GH5 (PaMan5A) and GH26 (PaMan26A) endo-β-1,4-mannanases from the coprophilic ascomycete Podospora anserina contribute to the enzymatic degradation of lignocellulosic biomass. In this study, P. anserina mannanases were further subjected to detailed comparative analysis of their substrate specificities, active site organization, and transglycosylation capacity. Although PaMan5A displays a classical mode of action, PaMan26A revealed an atypical hydrolysis pattern with the release of mannotetraose and mannose from mannopentaose resulting from a predominant binding mode involving the −4 subsite. The crystal structures of PaMan5A and PaMan26A were solved at 1.4 and 2.85 Å resolution, respectively. Analysis of the PaMan26A structure supported strong interaction with substrate at the −4 subsite mediated by two aromatic residues Trp-244 and Trp-245. The PaMan26A structure appended to its family 35 carbohydrate binding module revealed a short and proline-rich rigid linker that anchored together the catalytic and the binding modules. PMID:23558681

Biophysical insights into the interaction of clofazimine with human alpha 1-acid glycoprotein: a multitechnique approach.

PubMed

Ajmal, Mohammad Rehan; Almutairi, Fahad; Zaidi, Nida; Alam, Parvez; Siddiqi, Mohammad Khursheed; Khan, Mohsin Vahid; Zaman, Masihuz; Ishtikhar, Mohd; Khan, Rizwan Hasan

2018-04-25

Alpha1-acid glycoprotein (AAG) is a major acute phase protein of human plasma. Binding of clofazimine to AAG is investigated using optical spectroscopy and molecular docking tools. We found significant quenching of intrinsic fluorescence of AAG upon the binding of clofazimine, binding mode is static with binding constant of 3.52 × 10 4 at 298 K. The Gibbs free energy change is found to be negative for the interaction of clofazimine with AAG indicating spontaneity of the binding process. Binding of clofazimine induced ordered structure in protein and lead to molecular compaction. Molecular docking results indicate the binding site is located in the central beta barrel, hydrogen bonding and hydrophobic interactions are main bonding forces between AAG-clofazimine.

Exploring the binding pathways of the 14-3-3ζ protein: Structural and free-energy profiles revealed by Hamiltonian replica exchange molecular dynamics with distancefield distance restraints

PubMed Central

Nagy, Gabor; Oostenbrink, Chris; Hritz, Jozef

2017-01-01

The 14-3-3 protein family performs regulatory functions in eukaryotic organisms by binding to a large number of phosphorylated protein partners. Whilst the binding mode of the phosphopeptides within the primary 14-3-3 binding site is well established based on the crystal structures of their complexes, little is known about the binding process itself. We present a computational study of the process by which phosphopeptides bind to the 14-3-3ζ protein. Applying a novel scheme combining Hamiltonian replica exchange molecular dynamics and distancefield restraints allowed us to map and compare the most likely phosphopeptide-binding pathways to the 14-3-3ζ protein. The most important structural changes to the protein and peptides involved in the binding process were identified. In order to bind phosphopeptides to the primary interaction site, the 14-3-3ζ adopted a newly found wide-opened conformation. Based on our findings we additionally propose a secondary interaction site on the inner surface of the 14-3-3ζ dimer, and a direct interference on the binding process by the flexible C-terminal tail. A minimalistic model was designed to allow for the efficient calculation of absolute binding affinities. Binding affinities calculated from the potential of mean force along the binding pathway are in line with the available experimental estimates for two of the studied systems. PMID:28727767

Fine-tuning the extent and dynamics of binding cleft opening as a potential general regulatory mechanism in parvulin-type peptidyl prolyl isomerases

NASA Astrophysics Data System (ADS)

Czajlik, András; Kovács, Bertalan; Permi, Perttu; Gáspári, Zoltán

2017-03-01

Parvulins or rotamases form a distinct group within peptidyl prolyl cis-trans isomerases. Their exact mode of action as well as the role of conserved residues in the family are still not unambiguously resolved. Using backbone S2 order parameters and NOEs as restraints, we have generated dynamic structural ensembles of three distinct parvulins, SaPrsA, TbPin1 and CsPinA. The resulting ensembles are in good agreement with the experimental data but reveal important differences between the three enzymes. The largest difference can be attributed to the extent of the opening of the substrate binding cleft, along which motional mode the three molecules occupy distinct regions. Comparison with a wide range of other available parvulin structures highlights structural divergence along the bottom of the binding cleft acting as a hinge during the opening-closing motion. In the prototype WW-domain containing parvulin, Pin1, this region is also important in forming contacts with the WW domain known to modulate enzymatic activity of the catalytic domain. We hypothesize that modulation of the extent and dynamics of the identified ‘breathing motion’ might be one of the factors responsible for functional differences in the distinct parvulin subfamilies.

Comparing the conformational behavior of a series of diastereomeric cyclic urea HIV-1 inhibitors using the low mode:monte carlo conformational search method.

PubMed

Parish, Carol A; Yarger, Matthew; Sinclair, Kent; Dure, Myrianne; Goldberg, Alla

2004-09-23

The conformational flexibility of a series of diastereomeric cyclic urea HIV-1 protease inhibitors has been examined using the Low Mode:Monte Carlo conformational search method. Force fields were validated by a comparison of the energetic ordering of the minimum energy structures on the AMBER/GBSA(water), OPLSAA/GBSA(water) and HF/6-311G/SCRF(water) surfaces. The energetic ordering of the minima on the OPLSAA /GBSA(water) surface was in better agreement with the quantum calculations than the ordering on the AMBER/GBSA(water) surface. An ensemble of low energy structures was generated using OPLSAA/GBSA(water) and used to compare the molecular shape and flexibility of each diastereomer to the experimentally determined binding affinities and crystal structures of closely related systems. The results indicate that diastereomeric solution-phase energetic stability, conformational rigidity and ability to adopt a chair conformation correlate strongly with experimental binding affinities. Rigid body docking suggests that all of the diastereomers adopt solution-phase conformations suitable for alignment with the HIV-1 protease; however, these results indicate that the binding affinities are dependent upon subtle differences in the P1/P1' and P2/P2' substituent orientations.

The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly

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

Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk

The structure of a tubulin-binding cofactor from L. major is reported and compared with yeast, plant and human orthologues. Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are keymore » to association. This study provides a reagent and template to support further work in this area.« less

Design and synthesis of inositolphosphoglycan putative insulin mediators.

PubMed

López-Prados, Javier; Cuevas, Félix; Reichardt, Niels-Christian; de Paz, José-Luis; Morales, Ezequiel Q; Martín-Lomas, Manuel

2005-03-07

The binding modes of a series of molecules, containing the glucosamine (1-->6) myo-inositol structural motif, into the ATP binding site of the catalytic subunit of cAMP-dependent protein kinase (PKA) have been analysed using molecular docking. These calculations predict that the presence of a phosphate group at the non-reducing end in pseudodisaccharide and pseudotrisaccharide structures properly orientate the molecule into the binding site and that pseudotrisaccharide structures present the best shape complementarity. Therefore, pseudodisaccharides and pseudotrisaccharides have been synthesised from common intermediates using effective synthetic strategies. On the basis of this synthetic chemistry, the feasibility of constructing small pseudotrisaccharide libraries on solid-phase using the same intermediates has been explored. The results from the biological evaluation of these molecules provide additional support to an insulin-mediated signalling system which involves the intermediacy of inositolphosphoglycans as putative insulin mediators.

Investigate the Binding of Catechins to Trypsin Using Docking and Molecular Dynamics Simulation

PubMed Central

Cui, Fengchao; Yang, Kecheng; Li, Yunqi

2015-01-01

To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189–195, 214–220 and 225–228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas. PMID:25938485

Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: Advances toward treatment of hepatitis B virus infections

NASA Astrophysics Data System (ADS)

Shahabadi, Nahid; Falsafi, Monireh

The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33 ± 0.2 × 104 L mol-1and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH = 34.4 kJ mol-1; ΔS = 184.32 J mol-1 K-1). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol-1. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.

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

Crystal structures of ASK1-inhibtor complexes provide a platform for structure-based drug design

PubMed Central

Singh, Onkar; Shillings, Anthony; Craggs, Peter; Wall, Ian; Rowland, Paul; Skarzynski, Tadeusz; Hobbs, Clare I; Hardwick, Phil; Tanner, Rob; Blunt, Michelle; Witty, David R; Smith, Kathrine J

2013-01-01

ASK1, a member of the MAPK Kinase Kinase family of proteins has been shown to play a key role in cancer, neurodegeneration and cardiovascular diseases and is emerging as a possible drug target. Here we describe a ‘replacement-soaking’ method that has enabled the high-throughput X-ray structure determination of ASK1/ligand complexes. Comparison of the X-ray structures of five ASK1/ligand complexes from 3 different chemotypes illustrates that the ASK1 ATP binding site is able to accommodate a range of chemical diversity and different binding modes. The replacement-soaking system is also able to tolerate some protein flexibility. This crystal system provides a robust platform for ASK1/ligand structure determination and future structure based drug design. PMID:23776076

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction.

PubMed

Bule, Pedro; Pires, Virgínia M R; Alves, Victor D; Carvalho, Ana Luísa; Prates, José A M; Ferreira, Luís M A; Smith, Steven P; Gilbert, Harry J; Noach, Ilit; Bayer, Edward A; Najmudin, Shabir; Fontes, Carlos M G A

2018-05-03

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module.

PubMed

Wojciechowski, Michał; Różycki, Bartosz; Huy, Pham Dinh Quoc; Li, Mai Suan; Bayer, Edward A; Cieplak, Marek

2018-03-22

The assembly of the polysaccharide degradating cellulosome machinery is mediated by tight binding between cohesin and dockerin domains. We have used an empirical model known as FoldX as well as molecular mechanics methods to determine the free energy of binding between a cohesin and a dockerin from Clostridium thermocellum in two possible modes that differ by an approximately 180° rotation. Our studies suggest that the full-length wild-type complex exhibits dual binding at room temperature, i.e., the two modes of binding have comparable probabilities at equilibrium. The ability to bind in the two modes persists at elevated temperatures. However, single-point mutations or truncations of terminal segments in the dockerin result in shifting the equilibrium towards one of the binding modes. Our molecular dynamics simulations of mechanical stretching of the full-length wild-type cohesin-dockerin complex indicate that each mode of binding leads to two kinds of stretching pathways, which may be mistakenly taken as evidence of dual binding.

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

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.

The solution structure of the pentatricopeptide repeat protein PPR10 upon binding atpH RNA

PubMed Central

Gully, Benjamin S.; Cowieson, Nathan; Stanley, Will A.; Shearston, Kate; Small, Ian D.; Barkan, Alice; Bond, Charles S.

2015-01-01

The pentatricopeptide repeat (PPR) protein family is a large family of RNA-binding proteins that is characterized by tandem arrays of a degenerate 35-amino-acid motif which form an α-solenoid structure. PPR proteins influence the editing, splicing, translation and stability of specific RNAs in mitochondria and chloroplasts. Zea mays PPR10 is amongst the best studied PPR proteins, where sequence-specific binding to two RNA transcripts, atpH and psaJ, has been demonstrated to follow a recognition code where the identity of two amino acids per repeat determines the base-specificity. A recently solved ZmPPR10:psaJ complex crystal structure suggested a homodimeric complex with considerably fewer sequence-specific protein–RNA contacts than inferred previously. Here we describe the solution structure of the ZmPPR10:atpH complex using size-exclusion chromatography-coupled synchrotron small-angle X-ray scattering (SEC-SY-SAXS). Our results support prior evidence that PPR10 binds RNA as a monomer, and that it does so in a manner that is commensurate with a canonical and predictable RNA-binding mode across much of the RNA–protein interface. PMID:25609698

Identification of a Novel Class of BRD4 Inhibitors by Computational Screening and Binding Simulations

PubMed Central

2017-01-01

Computational screening is a method to prioritize small-molecule compounds based on the structural and biochemical attributes built from ligand and target information. Previously, we have developed a scalable virtual screening workflow to identify novel multitarget kinase/bromodomain inhibitors. In the current study, we identified several novel N-[3-(2-oxo-pyrrolidinyl)phenyl]-benzenesulfonamide derivatives that scored highly in our ensemble docking protocol. We quantified the binding affinity of these compounds for BRD4(BD1) biochemically and generated cocrystal structures, which were deposited in the Protein Data Bank. As the docking poses obtained in the virtual screening pipeline did not align with the experimental cocrystal structures, we evaluated the predictions of their precise binding modes by performing molecular dynamics (MD) simulations. The MD simulations closely reproduced the experimentally observed protein–ligand cocrystal binding conformations and interactions for all compounds. These results suggest a computational workflow to generate experimental-quality protein–ligand binding models, overcoming limitations of docking results due to receptor flexibility and incomplete sampling, as a useful starting point for the structure-based lead optimization of novel BRD4(BD1) inhibitors. PMID:28884163

Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

PubMed Central

Weber, Janine; Bao, Han; Hartlmüller, Christoph; Wang, Zhiqin; Windhager, Almut; Janowski, Robert; Madl, Tobias; Jin, Peng; Niessing, Dierk

2016-01-01

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases. DOI: http://dx.doi.org/10.7554/eLife.11297.001 PMID:26744780

The 3D Structure of the Binding Pocket of the Human Oxytocin Receptor for Benzoxazine Antagonists, Determined by Molecular Docking, Scoring Functions and 3D-QSAR Methods

NASA Astrophysics Data System (ADS)

Jójárt, Balázs; Martinek, Tamás A.; Márki, Árpád

2005-05-01

Molecular docking and 3D-QSAR studies were performed to determine the binding mode for a series of benzoxazine oxytocin antagonists taken from the literature. Structural hypotheses were generated by docking the most active molecule to the rigid receptor by means of AutoDock 3.05. The cluster analysis yielded seven possible binding conformations. These structures were refined by using constrained simulated annealing, and the further ligands were aligned in the refined receptor by molecular docking. A good correlation was found between the estimated Δ G bind and the p K i values for complex F. The Connolly-surface analysis, CoMFA and CoMSIA models q CoMFA 2 = 0.653, q CoMSA 2 = 0.630 and r pred,CoMFA 2 = 0.852 , r pred,CoMSIA 2 = 0.815) confirmed the scoring function results. The structural features of the receptor-ligand complex and the CoMFA and CoMSIA fields are in closely connected. These results suggest that receptor-ligand complex F is the most likely binding hypothesis for the studied benzoxazine analogs.

The Structural Basis for Recognition of the PreQ0 Metabolite by an Unusually Small Riboswitch Aptamer Domain*S⃞♦

PubMed Central

Spitale, Robert C.; Torelli, Andrew T.; Krucinska, Jolanta; Bandarian, Vahe; Wedekind, Joseph E.

2009-01-01

Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ1 riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ1 aptamer domain in complex with its precursor metabolite preQ0. The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated. PMID:19261617

Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

PubMed Central

Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

2014-01-01

The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

Solution structure of the Legionella pneumophila Mip-rapamycin complex.

PubMed

Ceymann, Andreas; Horstmann, Martin; Ehses, Philipp; Schweimer, Kristian; Paschke, Anne-Katrin; Steinert, Michael; Faber, Cornelius

2008-03-17

Legionella pneumphila is the causative agent of Legionnaires' disease. A major virulence factor of the pathogen is the homodimeric surface protein Mip. It shows peptidyl-prolyl cis/trans isomerase activty and is a receptor of FK506 and rapamycin, which both inhibit its enzymatic function. Insight into the binding process may be used for the design of novel Mip inhibitors as potential drugs against Legionnaires' disease. We have solved the solution structure of free Mip77-213 and the Mip77-213-rapamycin complex by NMR spectroscopy. Mip77-213 showed the typical FKBP-fold and only minor rearrangements upon binding of rapamycin. Apart from the configuration of a flexible hairpin loop, which is partly stabilized upon binding, the solution structure confirms the crystal structure. Comparisons to the structures of free FKBP12 and the FKBP12-rapamycin complex suggested an identical binding mode for both proteins. The structural similarity of the Mip-rapamycin and FKBP12-rapamycin complexes suggests that FKBP12 ligands may be promising starting points for the design of novel Mip inhibitors. The search for a novel drug against Legionnaires' disease may therefore benefit from the large variety of known FKBP12 inhibitors.

Solution structure of the Legionella pneumophila Mip-rapamycin complex

PubMed Central

Ceymann, Andreas; Horstmann, Martin; Ehses, Philipp; Schweimer, Kristian; Paschke, Anne-Katrin; Steinert, Michael; Faber, Cornelius

2008-01-01

Background Legionella pneumphila is the causative agent of Legionnaires' disease. A major virulence factor of the pathogen is the homodimeric surface protein Mip. It shows peptidyl-prolyl cis/trans isomerase activty and is a receptor of FK506 and rapamycin, which both inhibit its enzymatic function. Insight into the binding process may be used for the design of novel Mip inhibitors as potential drugs against Legionnaires' disease. Results We have solved the solution structure of free Mip77–213 and the Mip77–213-rapamycin complex by NMR spectroscopy. Mip77–213 showed the typical FKBP-fold and only minor rearrangements upon binding of rapamycin. Apart from the configuration of a flexible hairpin loop, which is partly stabilized upon binding, the solution structure confirms the crystal structure. Comparisons to the structures of free FKBP12 and the FKBP12-rapamycin complex suggested an identical binding mode for both proteins. Conclusion The structural similarity of the Mip-rapamycin and FKBP12-rapamycin complexes suggests that FKBP12 ligands may be promising starting points for the design of novel Mip inhibitors. The search for a novel drug against Legionnaires' disease may therefore benefit from the large variety of known FKBP12 inhibitors. PMID:18366641

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

PubMed

Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

1995-02-10

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

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

Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai

2012-02-21

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology ofmore » the ECD and a distinct mode of ligand recognition. Here we report a 1.9 {angstrom} crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.« less

Crystal Structure of an LSD-Bound Human Serotonin Receptor

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

Wacker, Daniel; Wang, Sheng; McCorvy, John D.

The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD’s key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR—a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD’s slow binding kinetics may be due to a “lid” formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatlymore » accelerates LSD’s binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD’s actions at human serotonin receptors.« less

Crystal Structure of an LSD-Bound Human Serotonin Receptor.

PubMed

Wacker, Daniel; Wang, Sheng; McCorvy, John D; Betz, Robin M; Venkatakrishnan, A J; Levit, Anat; Lansu, Katherine; Schools, Zachary L; Che, Tao; Nichols, David E; Shoichet, Brian K; Dror, Ron O; Roth, Bryan L

2017-01-26

The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT 2B . The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD's key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT 2B R and 5-HT 2A R-a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD's slow binding kinetics may be due to a "lid" formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD's binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD's actions at human serotonin receptors. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.

Solution structure and interactions of the Escherichia coli cell division activator protein CedA.

PubMed

Chen, Ho An; Simpson, Peter; Huyton, Trevor; Roper, David; Matthews, Stephen

2005-05-10

CedA is a protein that is postulated to be involved in the regulation of cell division in Escherichia coli and related organisms; however, little biological data about its possible mode of action are available. Here we present a three-dimensional structure of this protein as determined by NMR spectroscopy. The protein is made up of four antiparallel beta-strands, an alpha-helix, and a large unstructured stretch of residues at the N-terminus. It shows structural similarity to a family of DNA-binding proteins which interact with dsDNA via a three-stranded beta-sheet, suggesting that CedA may be a DNA-binding protein. The putative binding surface of CedA is predominantly positively charged with a number of basic residues surrounding a groove largely dominated by aromatic residues. NMR chemical shift perturbations and gel-shift experiments performed with CedA confirm that the protein binds dsDNA, and its interaction is mediated primarily via the beta-sheet.

An Exquisitely Specific PDZ/Target Recognition Revealed by the Structure of INAD PDZ3 in Complex with TRP Channel Tail.

PubMed

Ye, Fei; Liu, Wei; Shang, Yuan; Zhang, Mingjie

2016-03-01

The vast majority of PDZ domains are known to bind to a few C-terminal tail residues of target proteins with modest binding affinities and specificities. Such promiscuous PDZ/target interactions are not compatible with highly specific physiological functions of PDZ domain proteins and their targets. Here, we report an unexpected PDZ/target binding occurring between the scaffold protein inactivation no afterpotential D (INAD) and transient receptor potential (TRP) channel in Drosophila photoreceptors. The C-terminal 15 residues of TRP are required for the specific interaction with INAD PDZ3. The INAD PDZ3/TRP peptide complex structure reveals that only the extreme C-terminal Leu of TRP binds to the canonical αB/βB groove of INAD PDZ3. The rest of the TRP peptide, by forming a β hairpin structure, binds to a surface away from the αB/βB groove of PDZ3 and contributes to the majority of the binding energy. Thus, the INAD PDZ3/TRP channel interaction is exquisitely specific and represents a new mode of PDZ/target recognitions. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides

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

Pioszak, Augen A.; Parker, Naomi R.; Gardella, Thomas J.

2009-12-01

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic {alpha}-helix to the same hydrophobic groove in the ECD as occupied by PTH,more » but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally 'unwound.' The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.« less

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.

Computational study of the binding of CuII to Alzheimer's amyloid-beta peptide: do Abeta42 and Abeta40 bind copper in identical fashion?

PubMed

Mantri, Yogita; Fioroni, Marco; Baik, Mu-Hyun

2008-11-01

One of the many hypotheses on the pathogenesis of Alzheimer's disease is that the amyloid-beta peptide (Abeta) binds CuII and can catalytically generate H2O2, leading to oxidative damage in brain tissues. For a molecular level understanding of such catalysis it is critical to know the structure of the Abeta-CuII complex precisely. Unfortunately, no high-resolution structure is available to date and there is considerable debate over the copper coordination environment with no clear consensus on which residues are directly bound to CuII. Considering all plausible isomers of the copper-bound Abeta42 and Abeta40 using a combination of density functional theory and classical molecular dynamics methods, we report an atomic resolution structure for each possible complex. We evaluated the relative energies of these isomeric structures and surprisingly found that Abeta42 and Abeta40 display very different binding modes, suggesting that shorter peptides that are truncated at the C-terminus may not be realistic models for understanding the chemistry of the most neurotoxic peptide, Abeta42.

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

Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis

PubMed Central

Thiele, S; Mungalpara, J; Steen, A; Rosenkilde, M M; Våbenø, J

2014-01-01

Background and Purpose The cyclopentapeptide FC131 (cyclo(-L-Arg1-L-Arg2-L-2-Nal3-Gly4-D-Tyr5-)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4 have previously been suggested based on molecular docking guided by structure–activity relationship (SAR) data; however, none of these have been verified by in vitro experiments. Experimental Approach Heterologous 125I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data. Key Results The Arg2 and 2-Nal3 side chains of FC131 interact with residues in TM-3 (His113, Asp171) and TM-5 (hydrophobic pocket) respectively. Arg1 forms charge-charge interactions with Asp187 in ECL-2, while D-Tyr5 points to the extracellular side of CXCR4. Furthermore, the backbone of FC131 interacts with the chemokine receptor-conserved Glu288 via two water molecules. Intriguingly, Tyr116 and Glu288 form a H-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity. Conclusions and Implications Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes suggested from previous SAR studies. Furthermore, insights into the mechanism for CXCR4 activation by CXCL12 were gained. The combined findings will facilitate future design of novel CXCR4 antagonists. PMID:25039237

Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

PubMed

Lavoie, Mathieu; Abou Elela, Sherif

2008-08-19

Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

The Caenorhabditis elegans DAF-12 nuclear receptor: structure, dynamics, and interaction with ligands.

PubMed

Alvarez, Lautaro D; Mañez, Pau Arroyo; Estrin, Darío A; Burton, Gerardo

2012-07-01

A structure for the ligand binding domain (LBD) of the DAF-12 receptor from Caenorhabditis elegans was obtained from the X-ray crystal structure of the receptor LBD from Strongyloides stercoralis bound to (25R)-Δ(7)-dafachronic acid (DA) (pdb:3GYU). The model was constructed in the presence of the ligand using a combination of Modeller, Autodock, and molecular dynamics (MD) programs, and then its dynamical behavior was studied by MD. A strong ligand binding mode (LBM) was found, with the three arginines in the ligand binding pocket (LBP) contacting the C-26 carboxylate group of the DA. The quality of the ceDAF-12 model was then evaluated by constructing several ligand systems for which the experimental activity is known. Thus, the dynamical behavior of the ceDAF-12 complex with the more active (25S)-Δ(7)-DA showed two distinct binding modes, one of them being energetically more favorable compared with the 25R isomer. Then the effect of the Arg564Cys and Arg598Met mutations on the (25R)-Δ(7)-DA binding was analyzed. The MD simulations showed that in the first case the complex was unstable, consistent with the lack of transactivation activity of (25R)-Δ(7)-DA in this mutant. Instead, in the case of the Arg598Met mutant, known to produce a partial loss of activity, our model predicted smaller effects on the LBM with a more stable MD trajectory. The model also showed that removal of the C-25 methyl does not impede the simultaneous strong interaction of the carboxylate with the three arginines, predicting that 27-nor-DAs are putative ceDAF-12 ligands. Copyright © 2012 Wiley Periodicals, Inc.

Structure of Chlorobium tepidum sepiapterin reductase complex reveals the novel substrate binding mode for stereospecific production of L-threo-tetrahydrobiopterin.

PubMed

Supangat, Supangat; Seo, Kyung Hye; Choi, Yong Kee; Park, Young Shik; Son, Daeyoung; Han, Chang-deok; Lee, Kon Ho

2006-01-27

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.

The flexible C-terminal arm of the Lassa arenavirus Z-protein mediates interactions with multiple binding partners.

PubMed

May, Eric R; Armen, Roger S; Mannan, Aristotle M; Brooks, Charles L

2010-08-01

The arenavirus genome encodes for a Z-protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z-protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation, and apoptosis of infected cells. Using homology models of the Z-protein from Lassa strain arenavirus, replica exchange molecular dynamics (MD) was used to refine the structures, which were then subsequently clustered. Population-weighted ensembles of low-energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was identified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV-1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during MD trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C-terminal domain conformation of the most populated member of the representative ensemble shielded protein-binding recognition motifs for Tsg101 and eIF4E and represents the most populated state free in solution. We propose that C-terminal flexibility is key for mediating the different functional states of the Z-protein. (c) 2010 Wiley-Liss, Inc.

The Flexible C-terminal Arm of the Lassa Arenavirus Z-Protein Mediates Interactions with Multiple Binding Partners

PubMed Central

May, Eric R.; Armen, Roger S.; Mannan, Aristotle M.; Brooks, Charles L.

2010-01-01

The arenavirus genome encodes for a Z-protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z-protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation and apoptosis of infected cells. Using homology models of the Z-protein from Lassa strain arenavirus, replica exchange molecular dynamics were employed to refine the structures, which were then subsequently clustered. Population weighted ensembles of low energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was indentified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV-1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during molecular dynamics trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C-terminal domain conformation of the most populated member of the representative ensemble shielded protein binding recognition motifs for Tsg101 and eIF4E, and represents the most populated state free in solution. We propose that C-terminal flexibility is key for mediating the different functional states of the Z-protein. PMID:20544962

Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein, musashi1, as revealed by comparison of their structures, modes of interaction, surface electrostatic potentials, and backbone dynamics.

PubMed

Miyanoiri, Youhei; Kobayashi, Hisanori; Imai, Takao; Watanabe, Michinao; Nagata, Takashi; Uesugi, Seiichi; Okano, Hideyuki; Katahira, Masato

2003-10-17

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in maintenance of the character of progenitor cells. Musashi1 contains two ribonucleoprotein-type RNA-binding domains (RBDs), RBD1 and RBD2, the affinity to RNA of RBD1 being much higher than that of RBD2. We previously reported the structure and mode of interaction with RNA of RBD2. Here, we have determined the structure and mode of interaction with RNA of RBD1. We have also analyzed the surface electrostatic potential and backbone dynamics of both RBDs. The two RBDs exhibit the same ribo-nucleoprotein-type fold and commonly make contact with RNA on the beta-sheet side. On the other hand, there is a remarkable difference in surface electrostatic potential, the beta-sheet of RBD1 being positively charged, which is favorable for binding negatively charged RNA, but that of RBD2 being almost neutral. There is also a difference in backbone dynamics, the central portion of the beta-sheet of RBD1 being flexible, but that of RBD2 not being flexible. The flexibility of RBD1 may be utilized in the recognition process to facilitate an induced fit. Thus, comparative studies have revealed the origin of the higher affinity of RBD1 than that of RBD2 and indicated that the affinity of an RBD to RNA is not governed by its fold alone but is also determined by its surface electrostatic potential and/or backbone dynamics. The biological role of RBD2 with lower affinity is also discussed.

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

PubMed Central

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

2012-01-01

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

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.

Mixed-mode sorption of hydroxylated atrazine degradation products to sell: A mechanism for bound residue

USGS Publications Warehouse

Lerch, R.N.; Thurman, E.M.; Kruger, E.L.

1997-01-01

This study tested the hypothesis that sorption of hydroxylated atrazine degradation products (HADPs: hydroxyatrazine, HA; deethylhydroxyatrazine, DEHA; and deisopropylhydroxyatrazine, DIHA) to soils occurs by mixed-mode binding resulting from two simultaneous mechanisms: (1) cation exchange and (2) hydrophobic interaction. The objective was to use liquid chromatography and soil extraction experiments to show that mixed-mode binding is the mechanism controlling HADP sorption to soils and is also a mechanism for bound residue. Overall, HADP binding to solid-phase extraction (SPE) sorbents occurred in the order: cation exchange >> octadecyl (C18) >> cyanopropyl. Binding to cation exchange SPE and to a high-performance liquid chromatograph octyl (C8) column showed evidence for mixed-mode binding. Comparison of soil extracted by 0.5 M KH2P04, pH 7.5, or 25% aqueous CH3CN showed that, for HA and DIHA, cation exchange was a more important binding mechanism to soils than hydrophobic interaction. Based on differences between several extractants, the extent of HADP mixed-mode binding to soil occurred in the following order: HA > DIHA > DEHA. Mixed-mode extraction recovered 42.8% of bound atrazine residues from aged soil, and 88% of this fraction was identified as HADPs. Thus, a significant portion of bound atrazine residues in soils is sorbed by the mixed-mode binding mechanisms.

Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi *

PubMed Central

Jia, Min; Li, Jianchao; Zhu, Jinwei; Wen, Wenyu; Zhang, Mingjie; Wang, Wenning

2012-01-01

GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gαi/o during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gαi·GDP. The crystal structures of Gαi·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gαi interaction features when compared with the only high resolution structure known with GL/Gαi interaction between RGS14 and Gαi1. Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gαi·GDP. A highly conserved “double Arg finger” sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gαi. Together with the sequence alignment, we suggest that the LGN GL/Gαi interaction represents a general binding mode between GL motifs and Gαi. We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors. PMID:22952234

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box

DOE PAGES

Jia, Chuandong; Zuo, Wei; Yang, Dong; ...

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. Here, this similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives,more » 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.« less

Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

PubMed

Kyriakis, Efthimios; Stravodimos, George A; Kantsadi, Anastassia L; Chatzileontiadou, Demetra S M; Skamnaki, Vassiliki T; Leonidas, Demetres D

2015-07-08

We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 μM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

PubMed

Ha, Sookhee N; Hey, Pat J; Ransom, Rick W; Harrell, C Meacham; Murphy, Kathryn L; Chang, Ray; Chen, Tsing-Bau; Su, Dai-Shi; Markowitz, M Kristine; Bock, Mark G; Freidinger, Roger M; Hess, Fred J

2005-05-27

We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

Finding a Potential Dipeptidyl Peptidase-4 (DPP-4) Inhibitor for Type-2 Diabetes Treatment Based on Molecular Docking, Pharmacophore Generation, and Molecular Dynamics Simulation

PubMed Central

Meduru, Harika; Wang, Yeng-Tseng; Tsai, Jeffrey J. P.; Chen, Yu-Ching

2016-01-01

Dipeptidyl peptidase-4 (DPP-4) is the vital enzyme that is responsible for inactivating intestinal peptides glucagon like peptide-1 (GLP-1) and Gastric inhibitory polypeptide (GIP), which stimulates a decline in blood glucose levels. The aim of this study was to explore the inhibition activity of small molecule inhibitors to DPP-4 following a computational strategy based on docking studies and molecular dynamics simulations. The thorough docking protocol we applied allowed us to derive good correlation parameters between the predicted binding affinities (pKi) of the DPP-4 inhibitors and the experimental activity values (pIC50). Based on molecular docking receptor-ligand interactions, pharmacophore generation was carried out in order to identify the binding modes of structurally diverse compounds in the receptor active site. Consideration of the permanence and flexibility of DPP-4 inhibitor complexes by means of molecular dynamics (MD) simulation specified that the inhibitors maintained the binding mode observed in the docking study. The present study helps generate new information for further structural optimization and can influence the development of new DPP-4 inhibitors discoveries in the treatment of type-2 diabetes. PMID:27304951

Finding a Potential Dipeptidyl Peptidase-4 (DPP-4) Inhibitor for Type-2 Diabetes Treatment Based on Molecular Docking, Pharmacophore Generation, and Molecular Dynamics Simulation.

PubMed

Meduru, Harika; Wang, Yeng-Tseng; Tsai, Jeffrey J P; Chen, Yu-Ching

2016-06-13

Dipeptidyl peptidase-4 (DPP-4) is the vital enzyme that is responsible for inactivating intestinal peptides glucagon like peptide-1 (GLP-1) and Gastric inhibitory polypeptide (GIP), which stimulates a decline in blood glucose levels. The aim of this study was to explore the inhibition activity of small molecule inhibitors to DPP-4 following a computational strategy based on docking studies and molecular dynamics simulations. The thorough docking protocol we applied allowed us to derive good correlation parameters between the predicted binding affinities (pKi) of the DPP-4 inhibitors and the experimental activity values (pIC50). Based on molecular docking receptor-ligand interactions, pharmacophore generation was carried out in order to identify the binding modes of structurally diverse compounds in the receptor active site. Consideration of the permanence and flexibility of DPP-4 inhibitor complexes by means of molecular dynamics (MD) simulation specified that the inhibitors maintained the binding mode observed in the docking study. The present study helps generate new information for further structural optimization and can influence the development of new DPP-4 inhibitors discoveries in the treatment of type-2 diabetes.

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.

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.

Probing protein flexibility reveals a mechanism for selective promiscuity

PubMed Central

Pabon, Nicolas A; Camacho, Carlos J

2017-01-01

Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets. DOI: http://dx.doi.org/10.7554/eLife.22889.001 PMID:28432789

Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

PubMed Central

Chen, Jianzhong; Zhang, Dinglin; Zhang, Yuxin; Li, Guohui

2012-01-01

Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD) simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA) method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors. PMID:22408446

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.

PubMed Central

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases. PMID:11509371

Computational investigation of the binding mode of bis(hydroxylphenyl)arenes in 17β-HSD1: molecular dynamics simulations, MM-PBSA free energy calculations, and molecular electrostatic potential maps

NASA Astrophysics Data System (ADS)

Negri, Matthias; Recanatini, Maurizio; Hartmann, Rolf W.

2011-09-01

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the last step of the estrogen biosynthesis, namely the reduction of estrone to the biologically potent estradiol. As such it is a potentially attractive drug target for the treatment of estrogen-dependent diseases like breast cancer and endometriosis. 17β-HSD1 belongs to the bisubstrate enzymes and exists as an ensemble of conformations. These principally differ in the region of the βFαG'-loop, suggesting a prominent role in substrate and inhibitor binding. Although several classes of potent non-steroidal 17β-HSD1 inhibitors currently exist, their binding mode is still unclear. We aimed to elucidate the binding mode of bis(hydroxyphenyl)arenes, a highly potent class of 17β-HSD1 inhibitors, and to rank these compounds correctly with respect to their inhibitory potency, two essential aspects in drug design. Ensemble docking experiments resulted in a steroidal binding mode for the closed enzyme conformations and in an alternative mode for the opened and occluded conformers with the inhibitors placed below the NADPH interacting with it synergically via π-π stacking and H-bond formation. Both binding modes were investigated by MD simulations and MM-PBSA binding free energy estimations using as representative member for this class compound 1 (50 nM). Notably, only the alternative binding mode proved stable and was energetically more favorable, while when simulated in the steroidal binding mode compound 1 was displaced from the active site. In parallel, ab initio studies of small NADPH-inhibitor complexes were performed, which supported the importance of the synergistic interaction between inhibitors and cofactor.

Chloroquine Binding Reveals Flavin Redox Switch Function of Quinone Reductase 2*

PubMed Central

Leung, Kevin K. K.; Shilton, Brian H.

2013-01-01

Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P212121 to P21, with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4–5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe126–Leu136) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch. PMID:23471972

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

PubMed

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-10-31

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor*

PubMed Central

Hagen, Stefanie; Drepper, Friedel; Fischer, Sven; Fodor, Krisztian; Passon, Daniel; Platta, Harald W.; Zenn, Michael; Schliebs, Wolfgang; Girzalsky, Wolfgang; Wilmanns, Matthias; Warscheid, Bettina; Erdmann, Ralf

2015-01-01

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The KD value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo. PMID:26359497

Multimodal Imaging of Alzheimer Pathophysiology in the Brain's Default Mode Network

DOE PAGES

Shin, Jonghan; Kepe, Vladimir; Small, Gary W.; ...

2011-01-01

The spatial correlations between the brain's default mode network (DMN) and the brain regions known to develop pathophysiology in Alzheimer's disease (AD) have recently attracted much attention. In this paper, we compare results of different functional and structural imaging modalities, including MRI and PET, and highlight different patterns of anomalies observed within the DMN. Multitracer PET imaging in subjects with and without dementia has demonstrated that [C-11]PIB- and [F-18]FDDNP-binding patterns in patients with AD overlap within nodes of the brain's default network including the prefrontal, lateral parietal, lateral temporal, and posterior cingulate cortices, with the exception of the medial temporalmore » cortex (especially, the hippocampus) where significant discrepancy between increased [F-18]FDDNP binding and negligible [C-11]PIB-binding was observed. [F-18]FDDNP binding in the medial temporal cortex—a key constituent of the DMN—coincides with both the presence of amyloid and tau pathology, and also with cortical areas with maximal atrophy as demonstrated by T1-weighted MR imaging of AD patients.« less

Crystal structure of a minimal eIF4E–Cup complex reveals a general mechanism of eIF4E regulation in translational repression

PubMed Central

Kinkelin, Kerstin; Veith, Katharina; Grünwald, Marlene; Bono, Fulvia

2012-01-01

Cup is an eIF4E-binding protein (4E-BP) that plays a central role in translational regulation of localized mRNAs during early Drosophila development. In particular, Cup is required for repressing translation of the maternally contributed oskar, nanos, and gurken mRNAs, all of which are essential for embryonic body axis determination. Here, we present the 2.8 Å resolution crystal structure of a minimal eIF4E–Cup assembly, consisting of the interacting regions of the two proteins. In the structure, two separate segments of Cup contact two orthogonal faces of eIF4E. The eIF4E-binding consensus motif of Cup (YXXXXLΦ) binds the convex side of eIF4E similarly to the consensus of other eIF4E-binding proteins, such as 4E-BPs and eIF4G. The second, noncanonical, eIF4E-binding site of Cup binds laterally and perpendicularly to the eIF4E β-sheet. Mutations of Cup at this binding site were shown to reduce binding to eIF4E and to promote the destabilization of the associated mRNA. Comparison with the binding mode of eIF4G to eIF4E suggests that Cup and eIF4G binding would be mutually exclusive at both binding sites. This shows how a common molecular surface of eIF4E might recognize different proteins acting at different times in the same pathway. The structure provides insight into the mechanism by which Cup disrupts eIF4E–eIF4G interaction and has broader implications for understanding the role of 4E-BPs in translational regulation. PMID:22832024

Structural Characterization of an Alternative Mode of Tigecycline Binding to the Bacterial Ribosome

PubMed Central

Schedlbauer, Andreas; Kaminishi, Tatsuya; Ochoa-Lizarralde, Borja; Dhimole, Neha; Zhou, Shu; López-Alonso, Jorge P.

2015-01-01

Although both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in both in vitro and in vivo activity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to the tert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins. PMID:25753625

DNA as a Target for Anticancer Phen-Imidazole Pd(II) Complexes.

PubMed

Heydari, Maryam; Moghadam, Mahboube Eslami; Tarlani, AliAkbar; Farhangian, Hossein

2017-05-01

Imidazole ring is a known structure in many natural or synthetic drug molecules and its metal complexes can interact with DNA and do the cleavage. Hence, to study the influence of the structure and size of the ligand on biological behavior of metal complexes, two water-soluble Pd(II) complexes of phen and FIP ligands (where phen is 1,10-phenanthroline and FIP is 2-(Furan-2-yl)-1H-Imidazo[4,5-f][1, 10]phenanthroline) with the formula of [Pd(phen)(FIP)](NO 3 ) 2 and [Pd(FIP) 2 ]Cl 2 , that were activated against chronic myelogenous leukemia cell line, K562, were selected. Also, the interaction of these anticancer Pd(II) complexes with highly polymerized calf thymus DNA was extensively studied by means of electronic absorption, fluorescence, and circular dichroism in Tris-buffer. The results showed that the binding was positive cooperation and [Pd(phen)(FIP)](NO 3 ) 2 (K f  = 127 M -1 G = 1.2) exhibited higher binding constant and number of binding sites than [Pd(FIP) 2 ]Cl 2 (K f  = 13 M -1 G = 1.03) upon binding to DNA. The fluorescence data indicates that quenching effect for [Pd(phen)(FIP)](NO 3 ) 2 (K SV  = 58 mM -1 ) was higher than [Pd(FIP) 2 ]Cl 2 (K SV  = 12 mM -1 ). Also, [Pd(FIP) 2 ]Cl 2 interacts with ethidium bromide-DNA, as non-competitive inhibition, and can bind to DNA via groove binding and [Pd(phen)(FIP)](NO 3 ) 2 can intercalate in DNA. These results were confirmed by circular dichroism spectra. Docking data revealed that longer complexes have higher interaction energy and bind to DNA via groove binding. Graphical Abstract Two anticancer Pd(II) complexes of imidazole derivative have been synthesized and interacted with calf thymus DNA. Modes of binding have been studied by electronic absorption, fluorescence, and CD measurements. [Pd(FIP) 2 ]Cl 2 can bind to DNA via groove binding while intercalation mode of binding is observed for [Pd(phen)(FIP)](NO 3 ) 2 .

Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase

PubMed Central

Patra, Niladri; Ioannidis, Efthymios I.

2016-01-01

Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21–22 kcal/mol, in good agreement with experiment (18–19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are accessible, for example in other transferases. PMID:27564542

Structural studies of bovine, equine, and leporine serum albumin complexes with naproxen.

PubMed

Bujacz, Anna; Zielinski, Kamil; Sekula, Bartosz

2014-09-01

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein-ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA-NPS), equine (ESA-NPS), and leporine (LSA-NPS) determined to 2.58 Å (C2), 2.42 Å (P61), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA-NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA-NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. © 2014 Wiley Periodicals, Inc.

Structure and mechanism of the phage T4 recombination mediator protein UvsY

DOE PAGES

Gajewski, Stefan; Waddell, Michael Brett; Vaithiyalingam, Sivaraja; ...

2016-03-07

The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY–ssDNA interaction occurs within the assembly via twomore » distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNA–gp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNA-binding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA–UvsX filament.« less

Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

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

Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.

2010-11-05

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened ormore » eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.« 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.

Crystal Structure of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete chrysosporium*S⃞

PubMed Central

Ishida, Takuya; Fushinobu, Shinya; Kawai, Rie; Kitaoka, Motomitsu; Igarashi, Kiyohiko; Samejima, Masahiro

2009-01-01

Glycoside hydrolase family 55 consists of β-1,3-glucanases mainly from filamentous fungi. A β-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes β-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from β-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has two β-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two β-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various β-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the two β-helical domains, demonstrating an unexpected variety of carbohydrate binding modes. PMID:19193645

Biochemical and Structural Characterization of Bisubstrate Inhibitors of BasE, the Self-standing Non-Ribosomal Peptide Synthetase Adenylate-Forming Enzyme of Acinetobactin Synthesis†,‡

PubMed Central

Drake, Eric J.; Duckworth, Benjamin P.; Neres, João; Aldrich, Courtney C.; Gulick, Andrew M.

2010-01-01

The human pathogen Acinetobacter baumannii produces a siderophore called acinetobactin that is derived from one molecule each of threonine, histidine, and 2,3-dihydroxybenzoic acid (DHB). The activity of several non-ribosomal peptide synthetase (NRPS) enzymes is used to combine the building blocks into the final molecule. The acinetobactin synthesis pathway initiates with a self-standing adenylation enzyme, BasE, that activates the DHB molecule and covalently transfers it to the pantetheine cofactor of an aryl-carrier protein of BasF, a strategy that is shared with many siderophore-producing NRPS clusters. In this reaction, DHB reacts with ATP to form the aryl adenylate and pyrophosphate. In a second partial reaction, the DHB is transferred to the carrier protein. Inhibitors of BasE and related enzymes have been identified that prevent growth of bacteria on iron-limiting media. Recently, a new inhibitor of BasE has been identified via high-throughput screening using a fluorescence polarization displacement assay. We present here biochemical and structural studies to examine the binding mode of this inhibitor. The kinetics of the wild-type BasE enzyme is shown and inhibition studies demonstrate that the new compound exhibits competitive inhibition against both ATP and 2,3-dihydroxybenzoate. Structural examination of BasE bound to this inhibitor illustrates a novel binding mode in which the phenyl moiety partially fills the enzyme pantetheine binding tunnel. Structures of rationally designed bisubstrate inhibitors are also presented. PMID:20853905

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

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

Verebová, Valéria; Adamcik, Jozef; Danko, Patrik

2014-01-31

Highlights: • Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA. • Anthraquinones quinizarin and danthron lengthen linear DNA. • Anthraquinones quinizarin and danthron possess middle binding affinity to DNA. • Anthraquinones quinizarin and danthron interact with DNA by intercalating mode. - Abstract: The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone),more » with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.« less

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ.

PubMed

Dharmaiah, Srisathiyanarayanan; Bindu, Lakshman; Tran, Timothy H; Gillette, William K; Frank, Peter H; Ghirlando, Rodolfo; Nissley, Dwight V; Esposito, Dominic; McCormick, Frank; Stephen, Andrew G; Simanshu, Dhirendra K

2016-11-01

Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

Concentration-dependent Cu(II) binding to prion protein

NASA Astrophysics Data System (ADS)

Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

2008-03-01

The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

Dirac cones in isogonal hexagonal metallic structures

NASA Astrophysics Data System (ADS)

Wang, Kang

2018-03-01

A honeycomb hexagonal metallic lattice is equivalent to a triangular atomic one and cannot create Dirac cones in its electromagnetic wave spectrum. We study in this work the low-frequency electromagnetic band structures in isogonal hexagonal metallic lattices that are directly related to the honeycomb one and show that such structures can create Dirac cones. The band formation can be described by a tight-binding model that allows investigating, in terms of correlations between local resonance modes, the condition for the Dirac cones and the consequence of the third structure tile sustaining an extra resonance mode in the unit cell that induces band shifts and thus nonlinear deformation of the Dirac cones following the wave vectors departing from the Dirac points. We show further that, under structure deformation, the deformations of the Dirac cones result from two different correlation mechanisms, both reinforced by the lattice's metallic nature, which directly affects the resonance mode correlations. The isogonal structures provide new degrees of freedom for tuning the Dirac cones, allowing adjustment of the cone shape by modulating the structure tiles at the local scale without modifying the lattice periodicity and symmetry.

Molecular mechanics of Staphylococcus aureus adhesin, CNA, and the inhibition of bacterial adhesion by stretching collagen

PubMed Central

Madani, Ali; Garakani, Kiavash

2017-01-01

Bacterial adhesion to collagen, the most abundant protein in humans, is a critical step in the initiation and persistence of numerous bacterial infections. In this study, we explore the collagen binding mechanism of the multi-modular cell wall anchored collagen adhesin (CNA) in Staphylococcus aureus and examine how applied mechanical forces can modulate adhesion ability. The common structural-functional elements and domain organization of CNA are present across over 50 genera of bacteria. Through the use of molecular dynamics models and normal mode analysis, we shed light on the CNA’s structural and conformational dynamics and its interactions with collagen that lead to collagen binding. Our results suggest that the linker region, CNA165-173, acts as a hinge exhibiting bending, extensional, and torsional modes of structural flexibility and its residues are key in the interaction of the CNA-collagen complex. Steered molecular dynamics simulations were conducted with umbrella sampling. During the course of these simulations, the ‘locking’ latch from the CNA N2 domain was dissociated from its groove in the CNA N1 domain, implying the importance of the latch for effective ligand binding. Finally, we observed that the binding efficiency of the CNA N1-N2 domains to collagen decreases greatly with increasing tensile force application to the collagen peptides. Thus, CNA and similar adhesins might preferentially bind to sites in which collagen fibers are cleaved, such as in wounded, injured, or inflamed tissues, or in which the collagenous tissue is less mature. As alternative techniques for control of bacterial infection are in-demand due to the rise of bacterial antibiotic resistance, results from our computational studies with respect to the mechanoregulation of the collagen binding site may inspire new therapeutics and engineering solutions by mechanically preventing colonization and/or further pathogenesis. PMID:28665944

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.

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

PubMed

Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

1989-12-08

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.

Unveiling the Mode of Interaction of Berberine Alkaloid in Different Supramolecular Confined Environments: Interplay of Surface Charge between Nano-Confined Charged Layer and DNA.

PubMed

Kundu, Niloy; Roy, Arpita; Banik, Debasis; Sarkar, Nilmoni

2016-02-18

In this Article, we demonstrate a detailed characterization of binding interaction of berberine chloride (BBCl) with calf-thymus DNA (CT-DNA) in buffer solution as well as in two differently charged reverse micelles (RMs). The photophyscial properties of this alkaloid have been modulated within these microheterogeneous bioassemblies. The mode of binding of this alkaloid with DNA is of debate to date. However, fluorescence spectroscopic measurements, circular dichroism (CD) measurement, and temperature-dependent study unambiguously establish that BBCl partially intercalates into the DNA base pairs. The nonplanarity imposed by partial saturation in their structure causes the nonclassical types of intercalation into DNA. Besides the intercalation, electrostatic interactions also play a significant role in the binding between BBCl and DNA. DNA structure turns into a condensed form after encapsulation into RMs, which is followed by the CD spectra and microscopy study. The probe location and dynamics in the nanopool of the RMs depended on the electrostatic interaction between the charged surfactants and cationic berberine. The structural alteration of CT-DNA from B form to condensed form and the interplay of surface charge between RMs and DNA determine the interaction between the alkaloid and DNA in RMs. Time-resolved study and fluorescence anisotropy measurements successfully provide the binding interaction of BBCl in the nanopool of the RMs in the absence and in the presence of DNA. This study motivates us to judge further the potential applicability of this alkaloid in other biological systems or other biomimicking organized assemblies.

Structural analysis of a putative SAM-dependent methyltransferase, YtqB, from Bacillus subtilis.

PubMed

Park, Sun Cheol; Song, Wan Seok; Yoon, Sung-il

2014-04-18

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) methylate diverse biological molecules using a SAM cofactor. The ytqB gene of Bacillus subtilis encodes a putative MTase and its biological function has never been characterized. To reveal the structural features and the cofactor binding mode of YtqB, we have determined the crystal structures of YtqB alone and in complex with its cofactor, SAM, at 1.9 Å and 2.2 Å resolutions, respectively. YtqB folds into a β-sheet sandwiched by two α-helical layers, and assembles into a dimeric form. Each YtqB monomer contains one SAM binding site, which shapes SAM into a slightly curved conformation and exposes the reactive methyl group of SAM potentially to a substrate. Our comparative structural analysis of YtqB and its homologues indicates that YtqB is a SAM-dependent class I MTase, and provides insights into the substrate binding site of YtqB. Copyright © 2014 Elsevier Inc. All rights reserved.

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

DOE PAGES

Wallen, Jamie R.; Zhang, Hao; Weis, Caroline; ...

2017-01-03

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome

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

Wallen, Jamie R.; Zhang, Hao; Weis, Caroline

The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. then, the two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerasemore » binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. The collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications.« less

Binding properties of SUMO-interacting motifs (SIMs) in yeast.

PubMed

Jardin, Christophe; Horn, Anselm H C; Sticht, Heinrich

2015-03-01

Small ubiquitin-like modifier (SUMO) conjugation and interaction play an essential role in many cellular processes. A large number of yeast proteins is known to interact non-covalently with SUMO via short SUMO-interacting motifs (SIMs), but the structural details of this interaction are yet poorly characterized. In the present work, sequence analysis of a large dataset of 148 yeast SIMs revealed the existence of a hydrophobic core binding motif and a preference for acidic residues either within or adjacent to the core motif. Thus the sequence properties of yeast SIMs are highly similar to those described for human. Molecular dynamics simulations were performed to investigate the binding preferences for four representative SIM peptides differing in the number and distribution of acidic residues. Furthermore, the relative stability of two previously observed alternative binding orientations (parallel, antiparallel) was assessed. For all SIMs investigated, the antiparallel binding mode remained stable in the simulations and the SIMs were tightly bound via their hydrophobic core residues supplemented by polar interactions of the acidic residues. In contrary, the stability of the parallel binding mode is more dependent on the sequence features of the SIM motif like the number and position of acidic residues or the presence of additional adjacent interaction motifs. This information should be helpful to enhance the prediction of SIMs and their binding properties in different organisms to facilitate the reconstruction of the SUMO interactome.

Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: advances toward treatment of hepatitis B virus infections.

PubMed

Shahabadi, Nahid; Falsafi, Monireh

2014-05-05

The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33±0.2×10(4) L mol(-1)and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH=34.4 kJ mol(-1); ΔS=184.32 J mol(-1) K(-1)). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol(-1). This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

Biochemical and structural characterization of a novel cooperative binding mode by Pit-1 with CATT repeats in the macrophage migration inhibitory factor promoter

PubMed Central

Agarwal, Sorabh

2018-01-01

Abstract Overexpression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) is linked to a number of autoimmune diseases and cancer. MIF production has been correlated to the number of CATT repeats in a microsatellite region upstream of the MIF gene. We have characterized the interaction of pituitary-specific positive transcription factor 1 (Pit-1) with a portion of the MIF promoter region flanking a microsatellite polymorphism (−794 CATT5–8). Using fluorescence anisotropy, we quantified tight complex formation between Pit-1 and an oligonucleotide consisting of eight consecutive CATT repeats (8xCATT) with an apparent Kd of 35 nM. Using competition experiments we found a 23 base pair oligonucleotide with 4xCATT repeats to be the minimum DNA sequence necessary for high affinity interaction with Pit-1. The stoichiometry of the Pit-1 DNA interaction was determined to be 2:1 and binding is cooperative in nature. We subsequently structurally characterized the complex and discovered a completely novel binding mode for Pit-1 in contrast to previously described Pit-1 complex structures. The affinity of Pit-1 for the CATT target sequence was found to be highly dependent on cooperativity. This work lays the groundwork for understanding transcriptional regulation of MIF and pursuing Pit-1 as a therapeutic target to treat MIF-mediated inflammatory disorders. PMID:29186613

Ligand binding modes from low resolution GPCR models and mutagenesis: chicken bitter taste receptor as a test-case.

PubMed

Di Pizio, Antonella; Kruetzfeldt, Louisa-Marie; Cheled-Shoval, Shira; Meyerhof, Wolfgang; Behrens, Maik; Niv, Masha Y

2017-08-15

Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.

Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains

PubMed Central

Cronin, Thomas C; DiNitto, Jonathan P; Czech, Michael P; Lambright, David G

2004-01-01

The pleckstrin homology (PH) domains of the homologous proteins Grp1 (general receptor for phosphoinositides), ARNO (Arf nucleotide binding site opener), and Cytohesin-1 bind phosphatidylinositol (PtdIns) 3,4,5-trisphosphate with unusually high selectivity. Remarkably, splice variants that differ only by the insertion of a single glycine residue in the β1/β2 loop exhibit dual specificity for PtdIns(3,4,5)P3 and PtdIns(4,5)P2. The structural basis for this dramatic specificity switch is not apparent from the known modes of phosphoinositide recognition. Here, we report crystal structures for dual specificity variants of the Grp1 and ARNO PH domains in either the unliganded form or in complex with the head groups of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Loss of contacts with the β1/β2 loop with no significant change in head group orientation accounts for the significant decrease in PtdIns(3,4,5)P3 affinity observed for the dual specificity variants. Conversely, a small increase rather than decrease in affinity for PtdIns(4,5)P2 is explained by a novel binding mode, in which the glycine insertion alleviates unfavorable interactions with the β1/β2 loop. These observations are supported by a systematic mutational analysis of the determinants of phosphoinositide recognition. PMID:15359279

Mode localization in the cooperative dynamics of protein recognition

NASA Astrophysics Data System (ADS)

Copperman, J.; Guenza, M. G.

2016-07-01

The biological function of proteins is encoded in their structure and expressed through the mediation of their dynamics. This paper presents a study on the correlation between local fluctuations, binding, and biological function for two sample proteins, starting from the Langevin Equation for Protein Dynamics (LE4PD). The LE4PD is a microscopic and residue-specific coarse-grained approach to protein dynamics, which starts from the static structural ensemble of a protein and predicts the dynamics analytically. It has been shown to be accurate in its prediction of NMR relaxation experiments and Debye-Waller factors. The LE4PD is solved in a set of diffusive modes which span a vast range of time scales of the protein dynamics, and provides a detailed picture of the mode-dependent localization of the fluctuation as a function of the primary structure of the protein. To investigate the dynamics of protein complexes, the theory is implemented here to treat the coarse-grained dynamics of interacting macromolecules. As an example, calculations of the dynamics of monomeric and dimerized HIV protease and the free Insulin Growth Factor II Receptor (IGF2R) domain 11 and its IGF2R:IGF2 complex are presented. Either simulation-derived or experimentally measured NMR conformers are used as input structural ensembles to the theory. The picture that emerges suggests a dynamical heterogeneous protein where biologically active regions provide energetically comparable conformational states that are trapped by a reacting partner in agreement with the conformation-selection mechanism of binding.

Transition Metal Intercalators as Anticancer Agents—Recent Advances

PubMed Central

Deo, Krishant M.; Pages, Benjamin J.; Ang, Dale L.; Gordon, Christopher P.; Aldrich-Wright, Janice R.

2016-01-01

The diverse anticancer utility of cisplatin has stimulated significant interest in the development of additional platinum-based therapies, resulting in several analogues receiving clinical approval worldwide. However, due to structural and mechanistic similarities, the effectiveness of platinum-based therapies is countered by severe side-effects, narrow spectrum of activity and the development of resistance. Nonetheless, metal complexes offer unique characteristics and exceptional versatility, with the ability to alter their pharmacology through facile modifications of geometry and coordination number. This has prompted the search for metal-based complexes with distinctly different structural motifs and non-covalent modes of binding with a primary aim of circumventing current clinical limitations. This review discusses recent advances in platinum and other transition metal-based complexes with mechanisms of action involving intercalation. This mode of DNA binding is distinct from cisplatin and its derivatives. The metals focused on in this review include Pt, Ru and Cu along with examples of Au, Ni, Zn and Fe complexes; these complexes are capable of DNA intercalation and are highly biologically active. PMID:27809241

Binding mode dependent signaling for the detection of Cu2 +: An experimental and theoretical approach with practical applications

NASA Astrophysics Data System (ADS)

Ghosh, Soumen; Khan, Mehebub Ali; Ganguly, Aniruddha; Masum, Abdulla Al; Alam, Md. Akhtarul; Guchhait, Nikhil

2018-02-01

Two amido-schiff bases (3-Hydroxy-naphthalene-2-carboxylic acid pyren-1-ylmethylene-hydrazide and Naphthalene-2-carboxylic acid pyren-1-ylmethylene-hydrazide) have been synthesized having a common structural unit and only differs by a -OH group in the naphthalene ring. Both of them can detect Cu2 + ion selectively in semi-aqueous medium in distinctly different output modes (one detects Cu2 + by naked-eye color change where as the other detects Cu2 + by fluorescence enhancement). The difference in the binding of Cu 2 + with the compounds is the reason for this observation. The detection limit is found to be micromolar region for compound which contains -OH group whereas the compound without -OH group detects copper in nano-molar region. DFT calculations have been performed in order to demonstrate the structure of the compounds and their copper complexes. Practical utility has been explored by successful paper strip response of both the compounds. The biological applications have been evaluated in RAW 264.7.

A chitin deacetylase of Podospora anserina has two functional chitin binding domains and a unique mode of action.

PubMed

Hoßbach, Janina; Bußwinkel, Franziska; Kranz, Andreas; Wattjes, Jasper; Cord-Landwehr, Stefan; Moerschbacher, Bruno M

2018-03-01

Chitosan is a structurally diverse biopolymer that is commercially derived from chitin by chemical processing, but chitin deacetylases (CDAs) potentially offer a sustainable and more controllable approach allowing the production of chitosans with tailored structures and biological activities. We investigated the CDA from Podospora anserina (PaCDA) which is closely related to Colletotrichum lindemuthianum CDA in the catalytic domain, but unique in having two chitin-binding domains. We produced recombinant PaCDA in Hansenula polymorpha for biochemical characterization and found that the catalytic domain of PaCDA is also functionally similar to C. lindemuthianum CDA, though differing in detail. When studying the enzyme's mode of action on chitin oligomers by quantitative mass-spectrometric sequencing, we found almost all possible sequences up to full deacetylation but with a clear preference for specific products. Deletion muteins lacking one or both CBDs confirmed their proposed function in supporting the enzymatic conversion of the insoluble substrate colloidal chitin. Copyright © 2017. Published by Elsevier Ltd.

Synthesis, structure, DNA/BSA binding and antibacterial studies of NNO tridentate Schiff base metal complexes

NASA Astrophysics Data System (ADS)

Sakthi, Marimuthu; Ramu, Andy

2017-12-01

A new salicylaldehyde derived 2,4-diiodo-6-((2-phenylaminoethylimino)methyl)phenol Schiff base(L) and its transition metal complexes of the type MLCl where, M = Cu(II), Ni(II), Co(II), Mn(II) and Zn(II) have been synthesized. The coordination mode of Schiff base holding NNO donor atoms with metal ions was well investigated by elemental analysis, ESI-mass as well as IR, UV-vis, CV and NMR spectral studies. The binding efficiency and mode of these complexes with biological macromolecules viz., herring sperm DNA (HS- DNA) and bovine serum albumin (BSA) have been explored through various spectroscopic techniques. The characteristic changes in absorption, emission and, circular dichroism spectra of the complexes with DNA indicate the noticeable interaction between them. From the all spectral information complexes could interact with DNA via non-intercalation mode of binding. The hyperchromisim in absorption band and hypochromisim in emission intensity of BSA with different complex concentrations shown significant information, and the binding affinity value has been predicted from Stern-Volmer plots. Further, all the complexes could cleave the circular plasmid pUC19 DNA efficiently by using an activator H2O2. The ligand and all metal(II) complexes showed good antibacterial activities. The molecular docking studies of the complexes with DNA were performed in order to make a comparison and conclusion with spectral technic results.

Subatomic and atomic crystallographic studies of aldose reductase: implications for inhibitor binding.

PubMed

Podjarny, A; Cachau, R E; Schneider, T; Van Zandt, M; Joachimiak, A

2004-04-01

The determination of several of aldose reductase-inhibitor complexes at subatomic resolution has revealed new structural details, including the specific interatomic contacts involved in inhibitor binding. In this article, we review the structures of the complexes of ALR2 with IDD 594 (resolution: 0.66 angstrom, IC50 (concentration of the inhibitor that produced half-maximal effect): 30 nM, space group: P2(1)), IDD 393 (resolution: 0.90 angstrom, IC50: 6 nM, space group: P1), fidarestat (resolution: 0.92 angstrom, IC50: 9 nM, space group: P2(1)) and minalrestat (resolution: 1.10 angstrom, IC50: 73 nM, space group: P1). The structures are compared and found to be highly reproductible within the same space group (root mean square (RMS) deviations: 0.15 approximately 0.3 angstrom). The mode of binding of the carboxylate inhibitors IDD 594 and IDD 393 is analysed. The binding of the carboxylate head can be accurately determined by the subatomic resolution structures, since both the protonation states and the positions of the atoms are very precisely known. The differences appear in the binding in the specificity pocket. The high-resolution structures explain the differences in IC50, which are confirmed both experimentally by mass spectrometry measures of VC50 and theoretically by free energy perturbation calculations. The binding of the cyclic imide inhibitors fidarestat and minalrestat is also described, focusing on the observation of a Cl(-) ion which binds simultaneously with fidarestat. The presence of this anion, binding also to the active site residue His110, leads to a mechanism in which the inhibitor can bind in a neutral state and then become charged inside the active site pocket. This mechanism can explain the excellent in vivo properties of cyclic imide inhibitors. In summary, the complete and detailed information supplied by the subatomic resolution structures can explain the differences in binding energy of the different inhibitors.

Apo adenylate kinase encodes its holo form: a principal component and varimax analysis.

PubMed

Cukier, Robert I

2009-02-12

Adenylate kinase undergoes large-scale motions of its LID and AMP-binding (AMPbd) domains when its apo, open form closes over its substrates, AMP and Mg2+-ATP. It may be an example of an enzyme that provides an ensemble of conformations in its apo state from which its substrates can select and bind to produce catalytically competent conformations. In this work, the fluctuations of the enzyme apo Escherichia coli adenylate kinase (AKE) are obtained with molecular dynamics. The resulting trajectory is analyzed with principal component analysis (PCA) that decomposes the atom motions into orthogonal modes ordered by their decreasing contributions to the total protein fluctuation. In apo AKE, a small set of the PCA modes describes the bulk of the fluctuations. Identification of the atom motions that are important contributors to these modes is improved with the use of a varimax rotation method that rotates the PCA modes to a new mode set that concentrates the atom contributions to a smaller set of atoms in these new modes. In this way, the nature of the important motions of the LID and AMPbd domains are clarified. The dominant PCA modes are used to investigate if apo AKE can fluctuate to conformations that are holo-like, even though the apo trajectory is mainly confined to a region around the initial apo structure. This is accomplished by expressing the difference between the protein coordinates, obtained from the holo and apo crystal structures, using as a basis the PCA modes from the apo AKE trajectory. The coherent motion described by a small set of the apo PCA modes is shown to be able to produce protein conformations that are quite similar to the holo conformation of the protein. In this sense, apo AKE does encode in its fluctuations information about holo-like conformations.

Structure of tropinone reductase-II complexed with NADP+ and pseudotropine at 1.9 A resolution: implication for stereospecific substrate binding and catalysis.

PubMed

Yamashita, A; Kato, H; Wakatsuki, S; Tomizaki, T; Nakatsu, T; Nakajima, K; Hashimoto, T; Yamada, Y; Oda, J

1999-06-15

Tropinone reductase-II (TR-II) catalyzes the NADPH-dependent reduction of the carbonyl group of tropinone to a beta-hydroxyl group. The crystal structure of TR-II complexed with NADP+ and pseudotropine (psi-tropine) has been determined at 1.9 A resolution. A seven-residue peptide near the active site, disordered in the unliganded structure, is fixed in the ternary complex by participation of the cofactor and substrate binding. The psi-tropine molecule is bound in an orientation which satisfies the product configuration and the stereochemical arrangement toward the cofactor. The substrate binding site displays a complementarity to the bound substrate (psi-tropine) in its correct orientation. In addition, electrostatic interactions between the substrate and Glu156 seem to specify the binding position and orientation of the substrate. A comparison between the active sites in TR-II and TR-I shows that they provide different van der Waals surfaces and electrostatic features. These differences likely contribute to the correct binding mode of the substrates, which are in opposite orientations in TR-II and TR-I, and to different reaction stereospecificities. The active site structure in the TR-II ternary complex also suggests that the arrangement of the substrate, cofactor, and catalytic residues is stereoelectronically favorable for the reaction.

Discovery of a Kelch-like ECH-associated protein 1-inhibitory tetrapeptide and its structural characterization

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

Sogabe, Satoshi; Sakamoto, Kotaro; Kamada, Yusuke

Keap1 constitutively binds to the transcription factor Nrf2 to promote its degradation, resulting in negative modulation of genes involved in cellular protection against oxidative stress. Keap1 is increasingly recognized as an attractive target for treating diseases involving oxidative stress, including cancer, atherosclerosis, diabetes, arthritis, and neurodegeneration. We used phage-display peptide screening to identify a tetrapeptide showing moderate binding affinity, which inhibits the interaction between Nrf2 and Keap1. The tetrapeptide does not include an ETGE motif, which is a commonly found consensus sequence in known peptidic inhibitors. In addition to affinity parameters, IC{sub 50}, K{sub D}, and thermodynamic parameters, the crystalmore » structure of the complex was determined to elucidate the binding conformation. The binding interactions resemble those of known small-molecule inhibitors as opposed to those of substrates and peptidic inhibitors. Although the tetrapeptide's affinity is not very high, our results may help facilitate the designing of small-molecule inhibitors during lead generation in drug discovery. - Highlights: • Keap1 inhibitory tetrapeptide with moderate affinity was discovered. • Crystal structure of the complex showed the unique binding mode. • Structural information gives a valuable insight for design of therapeutic compounds.« less

Investigating the binding mechanism of novel 6-aminonicotinate-based antagonists with P2Y12 by 3D-QSAR, docking and molecular dynamics simulations.

PubMed

Zhou, Shengfu; Fang, Danqing; Tan, Shepei; Lin, Weicong; Wu, Wenjuan; Zheng, Kangcheng

2017-10-01

P2Y 12 receptor is an attractive target for the anti-platelet therapies, treating various thrombotic diseases. In this work, a total of 107 6-aminonicotinate-based compounds as potent P2Y 12 antagonists were studies by a molecular modeling study combining three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulations to explore the decisive binding conformations of these antagonists with P2Y 12 and the structural features for the activity. The optimum CoMFA and CoMSIA models identified satisfactory robustness and good predictive ability, with R 2  = .983, q 2  = .805, [Formula: see text] = .881 for CoMFA model, and R 2  = .935, q 2  = .762, [Formula: see text] = .690 for CoMSIA model, respectively. The probable binding modes of compounds and key amino acid residues were revealed by molecular docking. MD simulations and MM/GBSA free energy calculations were further performed to validate the rationality of docking results and to compare the binding modes of several compound pairs with different activities, and the key residues (Val102, Tyr105, Tyr109, His187, Val190, Asn191, Phe252, His253, Arg256, Tyr259, Thr260, Val279, and Lys280) for the higher activity were pointed out. The binding energy decomposition indicated that the hydrophobic and hydrogen bond interactions play important roles for the binding of compounds to P2Y 12 . We hope these results could be helpful in design of potent and selective P2Y 12 antagonists.

Insights into regioselective metabolism of mefenamic acid by cytochrome P450 BM3 mutants through crystallography, docking, molecular dynamics, and free energy calculations.

PubMed

Capoferri, Luigi; Leth, Rasmus; ter Haar, Ernst; Mohanty, Arun K; Grootenhuis, Peter D J; Vottero, Eduardo; Commandeur, Jan N M; Vermeulen, Nico P E; Jørgensen, Flemming Steen; Olsen, Lars; Geerke, Daan P

2016-03-01

Cytochrome P450 BM3 (CYP102A1) mutant M11 is able to metabolize a wide range of drugs and drug-like compounds. Among these, M11 was recently found to be able to catalyze formation of human metabolites of mefenamic acid and other nonsteroidal anti-inflammatory drugs (NSAIDs). Interestingly, single active-site mutations such as V87I were reported to invert regioselectivity in NSAID hydroxylation. In this work, we combine crystallography and molecular simulation to study the effect of single mutations on binding and regioselective metabolism of mefenamic acid by M11 mutants. The heme domain of the protein mutant M11 was expressed, purified, and crystallized, and its X-ray structure was used as template for modeling. A multistep approach was used that combines molecular docking, molecular dynamics (MD) simulation, and binding free-energy calculations to address protein flexibility. In this way, preferred binding modes that are consistent with oxidation at the experimentally observed sites of metabolism (SOMs) were identified. Whereas docking could not be used to retrospectively predict experimental trends in regioselectivity, we were able to rank binding modes in line with the preferred SOMs of mefenamic acid by M11 and its mutants by including protein flexibility and dynamics in free-energy computation. In addition, we could obtain structural insights into the change in regioselectivity of mefenamic acid hydroxylation due to single active-site mutations. Our findings confirm that use of MD and binding free-energy calculation is useful for studying biocatalysis in those cases in which enzyme binding is a critical event in determining the selective metabolism of a substrate. © 2016 Wiley Periodicals, Inc.

Improved estimation of ligand macromolecule binding affinities by linear response approach using a combination of multi-mode MD simulation and QM/MM methods

NASA Astrophysics Data System (ADS)

Khandelwal, Akash; Balaz, Stefan

2007-01-01

Structure-based predictions of binding affinities of ligands binding to proteins by coordination bonds with transition metals, covalent bonds, and bonds involving charge re-distributions are hindered by the absence of proper force fields. This shortcoming affects all methods which use force-field-based molecular simulation data on complex formation for affinity predictions. One of the most frequently used methods in this category is the Linear Response (LR) approach of Åquist, correlating binding affinities with van der Waals and electrostatic energies, as extended by Jorgensen's inclusion of solvent-accessible surface areas. All these terms represent the differences, upon binding, in the ensemble averages of pertinent quantities, obtained from molecular dynamics (MD) or Monte Carlo simulations of the complex and of single components. Here we report a modification of the LR approach by: (1) the replacement of the two energy terms through the single-point QM/MM energy of the time-averaged complex structure from an MD simulation; and (2) a rigorous consideration of multiple modes (mm) of binding. The first extension alleviates the force-field related problems, while the second extension deals with the ligands exhibiting large-scale motions in the course of an MD simulation. The second modification results in the correlation equation that is nonlinear in optimized coefficients, but does not lead to an increase in the number of optimized coefficients. The application of the resulting mm QM/MM LR approach to the inhibition of zinc-dependent gelatinase B (matrix metalloproteinase 9) by 28 hydroxamate ligands indicates a significant improvement of descriptive and predictive abilities.

Binding of resveratrol to the minor groove of DNA sequences with AATT and TTAA segments induces differential stability.

PubMed

Nair, Maya S; D'Mello, Samar; Pant, Rashmi; Poluri, Krishna Mohan

2017-05-01

Interactions of a natural stilbene compound, resveratrol with two DNA sequences containing AATT/TTAA segments have been studied. Resveratrol is found to interact with both the sequences. The mode of interaction has been studied using absorption, steady state fluorescence and circular dichroism spectroscopic techniques. UV-visible absorption and fluorescence studies provided the information regarding the binding constants and the stoichiometry of binding, whereas circular dichroism studies depicted the structural changes in DNA upon resveratrol binding. Our results evidenced that, though resveratrol showed similar affinity to both the sequences, the mode of interactions was different. The binding constants of resveratrol to AATT/TTAA sequences were found to be 7.55×10 5 M -1 and 5.42×10 5 M -1 respectively. Spectroscopic data evidenced for a groove binding interaction. Melting studies showed that the binding of resveratrol induces differential stability to the DNA sequences d(CGTTAACG) 2 and d(CGAATTCG) 2 . Fluorescence data showed a stoichiometry of 1:1 for d(CGAATTCG) 2 -resveratrol complex and 1:4 for d(CGTTAACG) 2 -resveratrol complex. Molecular docking studies demonstrated that resveratrol binds to the minor groove region of both the sequences to form stable complexes with varied atomic contacts to the DNA bases or backbone. Both the complexes are stabilized by hydrogen bond formation. Our results evidenced that modulation of DNA sequence within the same bases can greatly alter the binding geometry and stability of the complex upon binding to small molecule inhibitor compounds like resveratrol. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

PubMed

Henderson, Brittney R; Saeedi, Bejan J; Campagnola, Grace; Geiss, Brian J

2011-01-01

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Elastic Network Model of a Nuclear Transport Complex

NASA Astrophysics Data System (ADS)

Ryan, Patrick; Liu, Wing K.; Lee, Dockjin; Seo, Sangjae; Kim, Young-Jin; Kim, Moon K.

2010-05-01

The structure of Kap95p was obtained from the Protein Data Bank (www.pdb.org) and analyzed RanGTP plays an important role in both nuclear protein import and export cycles. In the nucleus, RanGTP releases macromolecular cargoes from importins and conversely facilitates cargo binding to exportins. Although the crystal structure of the nuclear import complex formed by importin Kap95p and RanGTP was recently identified, its molecular mechanism still remains unclear. To understand the relationship between structure and function of a nuclear transport complex, a structure-based mechanical model of Kap95p:RanGTP complex is introduced. In this model, a protein structure is simply modeled as an elastic network in which a set of coarse-grained point masses are connected by linear springs representing biochemical interactions at atomic level. Harmonic normal mode analysis (NMA) and anharmonic elastic network interpolation (ENI) are performed to predict the modes of vibrations and a feasible pathway between locked and unlocked conformations of Kap95p, respectively. Simulation results imply that the binding of RanGTP to Kap95p induces the release of the cargo in the nucleus as well as prevents any new cargo from attaching to the Kap95p:RanGTP complex.

Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallography studies on Aurora A interactions with VX-680 and with TPX2

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

Zhao, Baoguang; Smallwood, Angela; Yang, Jingsong

2008-10-24

VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 {angstrom} resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a {pi}-{pi} interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggestmore » that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.« less

Structural study of biologically significant ligands with major birch pollen allergen Betv1 by docking and molecular dynamics simulation

PubMed Central

Kundu, Sangeeta; Roy, Debjani

2010-01-01

The major birch pollen allergen, Betv1 of Betula verrucosa is the main causative agent of birch pollen allergy in humans. Betv1 is capable of binding several physiological ligands including fatty acids, flavones, cytokinins and sterols. Until now, no structural information from crystallography or NMR is available regarding binding mode of any of these ligands into the binding pocket of Betv1. In the present study thirteen ligands have been successfully docked into the hydrophobic cavity of Betv1 and binding free energies of the complexes have been calculated using AutoDock 3.0.5. A linear relationship with correlation coefficient (R2) of 0.6 is obtained between ΔGbs values plotted against their corresponding IC50 values. The complex formed between Betv1 and the best docking pose for each ligand has been optimized by molecular dynamics simulation. Here, we describe the ligand binding of Betv1, which provides insight into the biological function of this protein. This knowledge is required for structural alteration or inhibition of some of these ligands in order to modify the allergenic properties of this protein. PMID:20978606

A new structural framework for integrating replication protein A into DNA processing machinery

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

Brosey, Chris A; Yan, Chunli; Tsutakawa, Susan E

2013-01-01

By coupling the protection and organization of ssDNA with the recruitment and alignment of DNA processing factors, Replication Protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA manages to coordinate the biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA s DNA binding activity, combining small-angle x-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA s DNA-binding core. It has been long held that RPA engages ssDNA in three stages, but our data reveal that RPA undergoes two rather than threemore » transitions as it binds ssDNA. In contrast to previous models, RPA is more compact when fully engaged on 20-30 nucleotides of ssDNA than when DNA-free, and there is no evidence for significant population of a highly compacted structure in the initial 8-10 nucleotide binding mode. These results provide a new framework for understanding the integration of ssDNA into DNA processing machinery and how binding partners may manipulate RPA architecture to gain access to the substrate.« less

Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen-Manganese Transporter MntC

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

Gribenko, Alexey; Mosyak, Lidia; Ghosh, Sharmistha

MntC is a metal-binding protein component of the Mn 2 +-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensionalmore » structure of the protein was solved by X-ray crystallography at 2.2 Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn 2 +-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn 2 +-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium–hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn 2 +.« less

Synthesis, structure, DNA/protein binding, and cytotoxic activity of a rhodium(III) complex with 2,6-bis(2-benzimidazolyl)pyridine.

PubMed

Esteghamat-Panah, Roya; Hadadzadeh, Hassan; Farrokhpour, Hossein; Simpson, Jim; Abdolmaleki, Amir; Abyar, Fatemeh

2017-02-15

A new mononuclear rhodium(III) complex, [Rh(bzimpy)Cl 3 ] (bzimpy = 2,6-bis(2-benzimidazolyl)pyridine), was synthesized and characterized by elemental analysis and spectroscopic methods. The molecular structure of the complex was confirmed by single-crystal X-ray crystallography. The interaction of the complex with fish sperm DNA (FS-DNA) was investigated by UV spectroscopy, emission titration, and viscosity measurement in order to evaluate the possible DNA-binding mode and to calculate the corresponding DNA-binding constant. The results reveal that the Rh(III) complex interacts with DNA through groove binding mode with a binding affinity on the order of 10 4 . In addition, the binding of the Rh(III) complex to bovine serum albumin (BSA) was monitored by UV-Vis and fluorescence emission spectroscopy at different temperatures. The mechanism of the complex interaction was found to be static quenching. The thermodynamic parameters (ΔH, ΔS, and ΔG) obtained from the fluorescence spectroscopy data show that van der Waals interactions and hydrogen bonds play a major role in the binding of the Rh(III) complex to BSA. For the comparison of the DNA- and BSA-binding affinities of the free bzimpy ligand with its Rh(III) complex, the absorbance titration and fluorescence quenching experiments of the free bzimpy ligand with DNA and BSA were carried out. Competitive experiments using eosin Y and ibuprofen as site markers indicated that the complex was mainly located in the hydrophobic cavity of site I of the protein. These experimental results were confirmed by the results of molecular docking. Finally, the in vitro cytotoxicity properties of the Rh(III) complex against the MCF-7, K562, and HT-29 cell lines were evaluated and compared with those of the free ligand (bzimpy). It was found that the complexation process improved the anticancer activity significantly. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Structural basis for the binding of tryptophan-based motifs by δ-COP

PubMed Central

Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

2015-01-01

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy

PubMed Central

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-01-01

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicteddual binding modes across multiple bacterial species, our approach opens up newpossibilities for understanding assembly and catalytic properties of a broadrange of multi-enzyme complexes. DOI: http://dx.doi.org/10.7554/eLife.10319.001 PMID:26519733

Exploring the free-energy landscape of carbohydrate-protein complexes: development and validation of scoring functions considering the binding-site topology

NASA Astrophysics Data System (ADS)

Eid, Sameh; Saleh, Noureldin; Zalewski, Adam; Vedani, Angelo

2014-12-01

Carbohydrates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the structure-based design of carbohydrate-based ligands. We assembled a diverse data set comprising 273 carbohydrate-protein crystal structures with known binding affinity and evaluated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as combinations thereof. Unfortunately, the tested functions were not capable of reproducing binding affinities in the studied complexes. To simplify the complex free-energy surface of carbohydrate-protein systems, we classified the studied proteins according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classification scheme reproduced binding affinities in the carbohydrate data set with an r 2 of 0.71 and root-mean-squared-error of 1.25 kcal/mol ( N = 236). The improvement in model performance underlines the significance of the differences in the local micro-environments of carbohydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and solvent exposure.

BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain.

PubMed

Hagn, Franz; Klein, Christian; Demmer, Oliver; Marchenko, Natasha; Vaseva, Angelina; Moll, Ute M; Kessler, Horst

2010-01-29

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-mu, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

PubMed

Elgeti, Matthias; Kazmin, Roman; Rose, Alexander S; Szczepek, Michal; Hildebrand, Peter W; Bartl, Franz J; Scheerer, Patrick; Hofmann, Klaus Peter

2018-03-23

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G t α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A Comprehensive Docking and MM/GBSA Rescoring Study of Ligand Recognition upon Binding Antithrombin

DOE PAGES

Zhang, Xiaohua; Perez-Sanchez, Horacio; C. Lightstone, Felice

2017-04-06

A high-throughput virtual screening pipeline has been extended from single energetically minimized structure Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring to ensemble-average MM/GBSA rescoring. The correlation coefficient (R2) of calculated and experimental binding free energies for a series of antithrombin ligands has been improved from 0.36 to 0.69 when switching from the single-structure MM/GBSA rescoring to ensemble-average one. The electrostatic interactions in both solute and solvent are identified to play an important role in determining the binding free energy after the decomposition of the calculated binding free energy. Furthermore, the increasing negative charge of the compounds provides a more favorablemore » electrostatic energy change but creates a higher penalty for the solvation free energy. Such a penalty is compensated by the electrostatic energy change, which results in a better binding affinity. A highly hydrophobic ligand is determined by the docking program to be a non-specific binder. Finally, these results have demonstrated that it is very important to keep a few top poses for rescoring, if the binding is non-specific or the binding mode is not well determined by the docking calculation.« less

Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

NASA Astrophysics Data System (ADS)

Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

2013-12-01

The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

A Comprehensive Docking and MM/GBSA Rescoring Study of Ligand Recognition upon Binding Antithrombin

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

Zhang, Xiaohua; Perez-Sanchez, Horacio; C. Lightstone, Felice

A high-throughput virtual screening pipeline has been extended from single energetically minimized structure Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring to ensemble-average MM/GBSA rescoring. The correlation coefficient (R2) of calculated and experimental binding free energies for a series of antithrombin ligands has been improved from 0.36 to 0.69 when switching from the single-structure MM/GBSA rescoring to ensemble-average one. The electrostatic interactions in both solute and solvent are identified to play an important role in determining the binding free energy after the decomposition of the calculated binding free energy. Furthermore, the increasing negative charge of the compounds provides a more favorablemore » electrostatic energy change but creates a higher penalty for the solvation free energy. Such a penalty is compensated by the electrostatic energy change, which results in a better binding affinity. A highly hydrophobic ligand is determined by the docking program to be a non-specific binder. Finally, these results have demonstrated that it is very important to keep a few top poses for rescoring, if the binding is non-specific or the binding mode is not well determined by the docking calculation.« less

Structures of BIR domains from human NAIP and cIAP2.

PubMed

Herman, Maria Dolores; Moche, Martin; Flodin, Susanne; Welin, Martin; Trésaugues, Lionel; Johansson, Ida; Nilsson, Martina; Nordlund, Pär; Nyman, Tomas

2009-11-01

The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.

Structures of BIR domains from human NAIP and cIAP2

PubMed Central

Herman, Maria Dolores; Moche, Martin; Flodin, Susanne; Welin, Martin; Trésaugues, Lionel; Johansson, Ida; Nilsson, Martina; Nordlund, Pär; Nyman, Tomas

2009-01-01

The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1′–P4′ side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3′ position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2′ and P4′ pockets make similar interactions to those seen in other BIR domain–peptide complexes. The structures also reveal how a serine in the P1′ position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins. PMID:19923725

Kinetic, Thermodynamic, and Structural Characterizations of the Association between Nrf2-DLGex Degron and Keap1

PubMed Central

Fukutomi, Toshiaki; Takagi, Kenji; Mizushima, Tsunehiro; Ohuchi, Noriaki

2014-01-01

Transcription factor Nrf2 (NF-E2-related factor 2) coordinately regulates cytoprotective gene expression, but under unstressed conditions, Nrf2 is degraded rapidly through Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination. Nrf2 harbors two Keap1-binding motifs, DLG and ETGE. Interactions between these two motifs and Keap1 constitute a key regulatory nexus for cellular Nrf2 activity through the formation of a two-site binding hinge-and-latch mechanism. In this study, we determined the minimum Keap1-binding sequence of the DLG motif, the low-affinity latch site, and defined a new DLGex motif that covers a sequence much longer than that previously defined. We have successfully clarified the crystal structure of the Keap1-DC-DLGex complex at 1.6 Å. DLGex possesses a complicated helix structure, which interprets well the human-cancer-derived loss-of-function mutations in DLGex. In thermodynamic analyses, Keap1-DLGex binding is characterized as enthalpy and entropy driven, while Keap1-ETGE binding is characterized as purely enthalpy driven. In kinetic analyses, Keap1-DLGex binding follows a fast-association and fast-dissociation model, while Keap1-ETGE binding contains a slow-reaction step that leads to a stable conformation. These results demonstrate that the mode of DLGex binding to Keap1 is distinct from that of ETGE structurally, thermodynamically, and kinetically and support our contention that the DLGex motif serves as a converter transmitting environmental stress to Nrf2 induction as the latch site. PMID:24366543

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding.

PubMed

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su; Hille, Bertil

2017-07-11

Binding of agonists to G-protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M 1 muscarinic acetylcholine receptors (M 1 Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M 1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M 1 R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane.

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

PubMed Central

Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae; Koh, Duk-Su

2017-01-01

Binding of agonists to G-protein–coupled receptors (GPCRs) activates heterotrimeric G proteins and downstream signaling. Agonist-bound GPCRs are then phosphorylated by protein kinases and bound by arrestin to trigger desensitization and endocytosis. Arrestin plays another important signaling function. It recruits and regulates activity of an extracellular signal-regulated kinase (ERK) cascade. However, molecular details and timing of ERK activation remain fundamental unanswered questions that limit understanding of how arrestin-dependent GPCR signaling controls cell functions. Here we validate and model a system that tracks the dynamics of interactions of arrestin with receptors and of ERK activation using optical reporters. Our intermolecular FRET measurements in living cells are consistent with β-arrestin binding to M1 muscarinic acetylcholine receptors (M1Rs) in two different binding modes, transient and stable. The stable mode persists for minutes after agonist removal. The choice of mode is governed by phosphorylation on key residues in the third intracellular loop of the receptor. We detect a similar intramolecular conformational change in arrestin in either binding mode. It develops within seconds of arrestin binding to the M1 receptor, and it reverses within seconds of arrestin unbinding from the transient binding mode. Furthermore, we observed that, when stably bound to phosphorylated M1R, β-arrestin scaffolds and activates MEK-dependent ERK. In contrast, when transiently bound, β-arrestin reduces ERK activity via recruitment of a protein phosphatase. All this ERK signaling develops at the plasma membrane. In this scaffolding hypothesis, a shifting balance between the two arrestin binding modes determines the degree of ERK activation at the membrane. PMID:28652372

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin TransporterⓈ

PubMed Central

Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H.; Ecker, Gerhard F.; Freissmuth, Michael; Sucic, Sonja

2015-01-01

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

Structural basis for molecular recognition at serotonin receptors.

PubMed

Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Gao, Xiang; Zhou, X Edward; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L; Cherezov, Vadim; Stevens, Raymond C; Xu, H Eric

2013-05-03

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Structure based design of 11β-HSD1 inhibitors.

PubMed

Singh, Suresh; Tice, Colin

2010-11-01

Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11β-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

Template-Based Modeling of Protein-RNA Interactions.

PubMed

Zheng, Jinfang; Kundrotas, Petras J; Vakser, Ilya A; Liu, Shiyong

2016-09-01

Protein-RNA complexes formed by specific recognition between RNA and RNA-binding proteins play an important role in biological processes. More than a thousand of such proteins in human are curated and many novel RNA-binding proteins are to be discovered. Due to limitations of experimental approaches, computational techniques are needed for characterization of protein-RNA interactions. Although much progress has been made, adequate methodologies reliably providing atomic resolution structural details are still lacking. Although protein-RNA free docking approaches proved to be useful, in general, the template-based approaches provide higher quality of predictions. Templates are key to building a high quality model. Sequence/structure relationships were studied based on a representative set of binary protein-RNA complexes from PDB. Several approaches were tested for pairwise target/template alignment. The analysis revealed a transition point between random and correct binding modes. The results showed that structural alignment is better than sequence alignment in identifying good templates, suitable for generating protein-RNA complexes close to the native structure, and outperforms free docking, successfully predicting complexes where the free docking fails, including cases of significant conformational change upon binding. A template-based protein-RNA interaction modeling protocol PRIME was developed and benchmarked on a representative set of complexes.

Structural Basis of Semaphorin-Plexin Recognition and Viral Mimicry from Sema7A and A39R Complexes with PlexinC1

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

Liu, Heli; Juo, Z. Sean; Shim, Ann Hye-Ryong

Repulsive signaling by Semaphorins and Plexins is crucial for the development and homeostasis of the nervous, immune, and cardiovascular systems. Sema7A acts as both an immune and a neural Semaphorin through PlexinC1, and A39R is a Sema7A mimic secreted by smallpox virus. We report the structures of Sema7A and A39R complexed with the Semaphorin-binding module of PlexinC1. Both structures show two PlexinC1 molecules symmetrically bridged by Semaphorin dimers, in which the Semaphorin and PlexinC1 {beta} propellers interact in an edge-on, orthogonal orientation. Both binding interfaces are dominated by the insertion of the Semaphorin's 4c-4d loop into a deep groove inmore » blade 3 of the PlexinC1 propeller. A39R appears to achieve Sema7A mimicry by preserving key Plexin-binding determinants seen in the mammalian Sema7A complex that have evolved to achieve higher affinity binding to the host-derived PlexinC1. The complex structures support a conserved Semaphorin-Plexin recognition mode and suggest that Plexins are activated by dimerization.« less

Structural insights into the oligomerization mode of the human receptor for advanced glycation end-products.

PubMed

Yatime, Laure; Andersen, Gregers R

2013-12-01

The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor sensing endogenous stress signals associated with the development of various diseases, including diabetes, vascular complications, Alzheimer's disease and cancer. RAGE ligands include advanced glycation end-products, S100 proteins, high mobility group box 1 protein and amyloid β-peptides/fibrils. Their signalling through RAGE induces a sustained inflammation that accentuates tissue damage, thereby participating in disease progression. Receptor oligomerization appears to be a crucial parameter for the formation of active signalling complexes, although the precise mode of oligomerization remains unclear in the context of these various ligands. In the present study, we report the first crystal structure of the VC1C2 fragment of the RAGE ectodomain. This structure provides the first description of the C2 domain in the context of the entire ectodomain and supports the observation of its conformational freedom relative to the rigid VC1 domain tandem. In addition, we have obtained a new crystal structure of the RAGE VC1 fragment. The packing in both crystal structures reveals an association of the RAGE molecules through contacts between two V domains and the physiological relevance of this homodimerization mode is discussed. Based on homology with single-pass transmembrane receptors, we also suggest RAGE dimerization through a conserved GxxxG motif within its transmembrane domain. A multimodal homodimerization strategy of RAGE is proposed to form the structural basis for ligand-specific complex formation and signalling functions, as well as for RAGE-mediated cell adhesion. hRAGE_VC1C2 and hRAGE_VC1C2 bind by x-ray crystallography (View interaction) hRAGE_VC1 and hRAGE_VC1 bind by x-ray crystallography (View interaction). © 2013 FEBS.

Crystal Structure of Cockroach Allergen Bla g 2, an Unusual Zinc Binding Aspartic Protease with a Novel Mode of Self-inhibition

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

Gustchina, Alla; Li, Mi; Wunschmann, Sabina

2010-07-19

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity ofmore » the catalytic aspartate residues, increasing the distance between them to {approx}4 {angstrom} and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.« less

Crystal structure of Thermoplasma acidophilum XerA recombinase shows large C-shape clamp conformation and cis-cleavage mode for nucleophilic tyrosine.

PubMed

Jo, Chang Hwa; Kim, Junsoo; Han, Ah-reum; Park, Sam Yong; Hwang, Kwang Yeon; Nam, Ki Hyun

2016-03-01

Site-specific Xer recombination plays a pivotal role in reshuffling genetic information. Here, we report the 2.5 Å crystal structure of XerA from the archaean Thermoplasma acidophilum. Crystallographic data reveal a uniquely open conformational state, resulting in a C-shaped clamp with an angle of ~ 48° and a distance of 57 Å between the core-binding and the catalytic domains. The catalytic nucleophile, Tyr264, is positioned in cis-cleavage mode by XerA's C-term tail that interacts with the CAT domain of a neighboring monomer without DNA substrate. Structural comparisons of tyrosine recombinases elucidate the dynamics of Xer recombinase. © 2016 Federation of European Biochemical Societies.

Alternative modes of client binding enable functional plasticity of Hsp70

NASA Astrophysics Data System (ADS)

Mashaghi, Alireza; Bezrukavnikov, Sergey; Minde, David P.; Wentink, Anne S.; Kityk, Roman; Zachmann-Brand, Beate; Mayer, Matthias P.; Kramer, Günter; Bukau, Bernd; Tans, Sander J.

2016-11-01

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Structure-Activity Relationships of the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PF-46396.

PubMed

Murgatroyd, Christopher; Pirrie, Lisa; Tran, Fanny; Smith, Terry K; Westwood, Nicholas J; Adamson, Catherine S

2016-09-15

HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. The lack of high-resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor-binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396 as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid-derived maturation inhibitors; therefore, our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by similar modes of action. Copyright © 2016 Murgatroyd et al.

NMR structure and dynamics of the engineered fluorescein-binding lipocalin FluA reveal rigidification of beta-barrel and variable loops upon enthalpy-driven ligand binding.

PubMed

Mills, Jeffrey L; Liu, Gaohua; Skerra, Arne; Szyperski, Thomas

2009-08-11

The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel beta-strands forming a beta-barrel with an alpha-helix attached to its side. Comparison of the NMR structure of free FluA with the X-ray structures of BBP.biliverdin IX(gamma) and FluA.fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the beta-barrel as well as adjoining beta-strand segments. An "induced fit" became apparent for the side chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse (15)N backbone spin relaxation times in the rotating frame for free FluA and also for the FluA.fluorescein complex. A reduction in the level of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy-driven, thus overcompensating for the negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction not only provides insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but also supports the future design of corresponding binding proteins with novel specificities, so-called "anticalins".

Comparison of crystal structures of human type 3 3α-hydroxysteroid dehydrogenase reveals an “induced-fit” mechanism and a conserved basic motif involved in the binding of androgen

PubMed Central

Couture, Jean-François; Pereira De Jésus-Tran, Karine; Roy, Anne-Marie; Cantin, Line; Côté, Pierre-Luc; Legrand, Pierre; Luu-The, Van; Labrie, Fernand; Breton, Rock

2005-01-01

The aldo-keto reductase (AKR) human type 3 3α-hydroxysteroid dehydrogenase (h3α–HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5α-dihydrotestosterone (5α-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3α–HSD3-NADP(H) at 1.9 Å resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3α–HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of themobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 Å3 in the presence of testosterone/acetate to 704 Å3 in the acetate/citratecomplex), are reminiscent of the “induced-fit” mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg301 and Arg304, localized near the steroid-binding cavity, significantly affects the 3α–HSD activity of this enzyme toward 5α-DHT and completely abolishes its 17β–HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens. PMID:15929998

Synthesis, characterization, crystal structure and DNA-binding studies of transition metal hydrazone complexes

NASA Astrophysics Data System (ADS)

Kanchanadevi, S.; Parveen, S.; Mahalingam, V.

2018-04-01

Three new complexes containing salicylaldazine (HL) ligand were synthesised by reacting suitable precursor complex [MCl2(PPh3)2] with the ligand (where M = Cu(II) or Ni(II) or Co(II)). The new complexes were characterised by various spectral studies such as IR, UV-Vis,1H NMR,EPR,fluorescence and elemental analyses. The binding modes of the complexes with HS-DNA have been studied by UV-Vis absorption titration. Binding of the complexes with bovine serum albumin (BSA) protein has been investigated using UV-visible, fluorescence and synchronous fluorescence spectroscopic methods. Redox behaviour of the complexes has been investigated by cyclic voltammetry.

Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations

PubMed Central

Moraca, Federica; Amato, Jussara; Ortuso, Francesco; Artese, Anna; Novellino, Ettore; Alcaro, Stefano; Parrinello, Michele; Limongelli, Vittorio

2017-01-01

G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 (d[AG3(T2AG3)3]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy (ΔGb0 = −10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands. PMID:28232513

General molecular mechanics method for transition metal carboxylates and its application to the multiple coordination modes in mono- and dinuclear Mn(II) complexes.

PubMed

Deeth, Robert J

2008-08-04

A general molecular mechanics method is presented for modeling the symmetric bidentate, asymmetric bidentate, and bridging modes of metal-carboxylates with a single parameter set by using a double-minimum M-O-C angle-bending potential. The method is implemented within the Molecular Operating Environment (MOE) with parameters based on the Merck molecular force field although, with suitable modifications, other MM packages and force fields could easily be used. Parameters for high-spin d (5) manganese(II) bound to carboxylate and water plus amine, pyridyl, imidazolyl, and pyrazolyl donors are developed based on 26 mononuclear and 29 dinuclear crystallographically characterized complexes. The average rmsd for Mn-L distances is 0.08 A, which is comparable to the experimental uncertainty required to cover multiple binding modes, and the average rmsd in heavy atom positions is around 0.5 A. In all cases, whatever binding mode is reported is also computed to be a stable local minimum. In addition, the structure-based parametrization implicitly captures the energetics and gives the same relative energies of symmetric and asymmetric coordination modes as density functional theory calculations in model and "real" complexes. Molecular dynamics simulations show that carboxylate rotation is favored over "flipping" while a stochastic search algorithm is described for randomly searching conformational space. The model reproduces Mn-Mn distances in dinuclear systems especially accurately, and this feature is employed to illustrate how MM calculations on models for the dimanganese active site of methionine aminopeptidase can help determine some of the details which may be missing from the experimental structure.

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein*

PubMed Central

Vlach, Jiri; Samal, Alexandra B.; Saad, Jamil S.

2014-01-01

Subcellular distribution of calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM co-localizes and interacts with the HIV-1 Gag protein in the cytosol of infected cells. Although it has been shown that binding of Gag to CaM is mediated by the matrix (MA) domain, the structural details of this interaction are not known. We have recently shown that binding of CaM to MA induces a conformational change that triggers myristate exposure, and that the CaM-binding domain of MA is confined to a region spanning residues 8–43 (MA-(8–43)). Here, we present the NMR structure of CaM bound to MA-(8–43). Our data revealed that MA-(8–43), which contains a novel CaM-binding motif, binds to CaM in an antiparallel mode with the N-terminal helix (α1) anchored to the CaM C-terminal lobe, and the C-terminal helix (α2) of MA-(8–43) bound to the N-terminal lobe of CaM. The CaM protein preserves a semiextended conformation. Binding of MA-(8–43) to CaM is mediated by numerous hydrophobic interactions and stabilized by favorable electrostatic contacts. Our structural data are consistent with the findings that CaM induces unfolding of the MA protein to have access to helices α1 and α2. It is noteworthy that several MA residues involved in CaM binding have been previously implicated in membrane binding, envelope incorporation, and particle production. The present findings may ultimately help in identification of the functional role of CaM in HIV-1 replication. PMID:24500712

BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

PubMed

Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris

2016-10-01

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

PubMed

Amano, Yasushi; Tanabe, Eiki; Yamaguchi, Tomohiko

2015-05-15

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800μM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51μM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural Analysis of HMGD-DNA Complexes Reveal Influence of Intercalation on Sequence Selectivity and DNA Bending

PubMed Central

Churchill, Mair E.A.; Klass, Janet; Zoetewey, David L.

2010-01-01

The ubiquitous eukaryotic High-Mobility-Group-Box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and fifty base pairs of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1-boxA-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending. PMID:20800069

Investigation of the bindings of a class of inhibitors with GSK3β kinase using thermodynamic integration MD simulation and kinase assay.

PubMed

Hsu, Chia-Jen; Hsu, Wen-Chi; Lee, Der-Jay; Liu, An-Lun; Chang, Chia-Ming; Shih, Huei-Jhen; Huang, Wun-Han; Lee-Chen, Guey-Jen; Hsieh-Li, Hsiu Mei; Lee, Guan-Chiun; Sun, Ying-Chieh

2017-08-01

GSK3β kinase is a noteworthy target for discovery of the drugs that will be used to treat several diseases. In the effort to identify a new inhibitor lead compound, we utilized thermodynamic integration (TI)-molecular dynamics (MD) simulation and kinase assay to investigate the bindings between GSK3β kinase and five compounds that were analogous to a known inhibitor with an available crystal structure. TI-MD simulations of the first two compounds (analogs 1 and 2) were used for calibration. The computed binding affinities of analogs 1 and 2 agreed well with the experimental results. The rest three compounds (analogs 3-5) were newly obtained from a database search, and their affinity data were newly measured in our labs. TI-MD simulations predicted the binding modes and the computed ΔΔG values have a reasonably good correlation with the experimental affinity data. These newly identified inhibitors appear to be new leads according to our survey of GSK3β inhibitors listed in recent review articles. The predicted binding modes of these compounds should aid in designing new derivatives of these compounds in the future. © 2017 John Wiley & Sons A/S.

Dual thio-digalactoside-binding modes of human galectins as the structural basis for the design of potent and selective inhibitors

PubMed Central

Hsieh, Tung-Ju; Lin, Hsien-Ya; Tu, Zhijay; Lin, Ting-Chien; Wu, Shang-Chuen; Tseng, Yu-Yao; Liu, Fu-Tong; Hsu, Shang-Te Danny; Lin, Chun-Hung

2016-01-01

Human galectins are promising targets for cancer immunotherapeutic and fibrotic disease-related drugs. We report herein the binding interactions of three thio-digalactosides (TDGs) including TDG itself, TD139 (3,3’-deoxy-3,3’-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside, recently approved for the treatment of idiopathic pulmonary fibrosis), and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) with human galectins-1, -3 and -7 as assessed by X-ray crystallography, isothermal titration calorimetry and NMR spectroscopy. Five binding subsites (A–E) make up the carbohydrate-recognition domains of these galectins. We identified novel interactions between an arginine within subsite E of the galectins and an arene group in the ligands. In addition to the interactions contributed by the galactosyl sugar residues bound at subsites C and D, the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterised dual binding modes demonstrate how binding potency, reported as decreased Kd values of the TDG inhibitors from μM to nM, is improved and also offer insights to development of selective inhibitors for individual galectins. PMID:27416897

TIA-1 RRM23 binding and recognition of target oligonucleotides

PubMed Central

Waris, Saboora; García-Mauriño, Sofía M.; Sivakumaran, Andrew; Beckham, Simone A.; Loughlin, Fionna E.; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C.J.

2017-01-01

Abstract TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. PMID:28184449

TIA-1 RRM23 binding and recognition of target oligonucleotides.

PubMed

Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

2017-05-05

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

How to deal with multiple binding poses in alchemical relative protein-ligand binding free energy calculations.

PubMed

Kaus, Joseph W; Harder, Edward; Lin, Teng; Abel, Robert; McCammon, J Andrew; Wang, Lingle

2015-06-09

Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the ligands. This improved the root-mean-square error (RMSE) for the predicted binding free energy from 1.9 kcal/mol with the original partial charges to 1.3 kcal/mol with the corrected partial charges.

How To Deal with Multiple Binding Poses in Alchemical Relative Protein–Ligand Binding Free Energy Calculations

PubMed Central

2016-01-01

Recent advances in improved force fields and sampling methods have made it possible for the accurate calculation of protein–ligand binding free energies. Alchemical free energy perturbation (FEP) using an explicit solvent model is one of the most rigorous methods to calculate relative binding free energies. However, for cases where there are high energy barriers separating the relevant conformations that are important for ligand binding, the calculated free energy may depend on the initial conformation used in the simulation due to the lack of complete sampling of all the important regions in phase space. This is particularly true for ligands with multiple possible binding modes separated by high energy barriers, making it difficult to sample all relevant binding modes even with modern enhanced sampling methods. In this paper, we apply a previously developed method that provides a corrected binding free energy for ligands with multiple binding modes by combining the free energy results from multiple alchemical FEP calculations starting from all enumerated poses, and the results are compared with Glide docking and MM-GBSA calculations. From these calculations, the dominant ligand binding mode can also be predicted. We apply this method to a series of ligands that bind to c-Jun N-terminal kinase-1 (JNK1) and obtain improved free energy results. The dominant ligand binding modes predicted by this method agree with the available crystallography, while both Glide docking and MM-GBSA calculations incorrectly predict the binding modes for some ligands. The method also helps separate the force field error from the ligand sampling error, such that deviations in the predicted binding free energy from the experimental values likely indicate possible inaccuracies in the force field. An error in the force field for a subset of the ligands studied was identified using this method, and improved free energy results were obtained by correcting the partial charges assigned to the ligands. This improved the root-mean-square error (RMSE) for the predicted binding free energy from 1.9 kcal/mol with the original partial charges to 1.3 kcal/mol with the corrected partial charges. PMID:26085821

Interaction of Sulforaphane with DNA and RNA

PubMed Central

Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

2015-01-01

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

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.

Why double-stranded RNA resists condensation

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

Tolokh, Igor S.; Pabit, Suzette; Katz, Andrea M.

2014-09-15

The addition of small amounts of multivalent cations to solutions containing double-stranded DNA leads to attraction between the negatively charged helices and eventually to condensation. Surprisingly, this effect is suppressed in double-stranded RNA, which carries the same charge as the DNA, but assumes a different double helical form. However, additional characterization of short (25 base-pairs) nucleic acid (NA) duplex structures by circular dichroism shows that measured differences in condensation are not solely determined by duplex helical geometry. Here we combine experiment, theory, and atomistic simulations to propose a mechanism that connects the observed variations in condensation of short NA duplexesmore » with the spatial variation of cobalt hexammine (CoHex) binding at the NA duplex surface. The atomistic picture that emerged showed that CoHex distributions around the NA reveals two major NA-CoHex binding modes -- internal and external -- distinguished by the proximity of bound CoHex to the helical axis. Decreasing trends in experimentally observed condensation propensity of the four studied NA duplexes (from B-like form of homopolymeric DNA, to mixed sequence DNA, to DNA:RNA hybrid, to A-like RNA) are explained by the progressive decrease of a single quantity: the fraction of CoHex ions in the external binding mode. Thus, while NA condensation depends on a complex interplay between various structural and sequence features, our coupled experimental and theoretical results suggest a new model in which a single parameter connects the NA condensation propensity with geometry and sequence dependence of CoHex binding.« less

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

PubMed Central

Kuznetsova, Irina M.; Sulatskaya, Anna I.; Uversky, Vladimir N.; Turoverov, Konstantin K.

2012-01-01

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates. PMID:22383971

Analyzing thioflavin T binding to amyloid fibrils by an equilibrium microdialysis-based technique.

PubMed

Kuznetsova, Irina M; Sulatskaya, Anna I; Uversky, Vladimir N; Turoverov, Konstantin K

2012-01-01

A new approach for the determination of the amyloid fibril - thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates.

Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors.

PubMed

Kast, David J; Yang, Changsong; Disanza, Andrea; Boczkowska, Malgorzata; Madasu, Yadaiah; Scita, Giorgio; Svitkina, Tatyana; Dominguez, Roberto

2014-04-01

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

Structural Probing of Off-Target G Protein-Coupled Receptor Activities within a Series of Adenosine/Adenine Congeners

PubMed Central

Paoletta, Silvia; Tosh, Dilip K.; Salvemini, Daniela; Jacobson, Kenneth A.

2014-01-01

We studied patterns of off-target receptor interactions, mostly at G protein-coupled receptors (GPCRs) in the µM range, of nucleoside derivatives that are highly engineered for nM interaction with adenosine receptors (ARs). Because of the considerable interest of using AR ligands for treating diseases of the CNS, we used the Psychoactive Drug Screening Program (PDSP) for probing promiscuity of these adenosine/adenine congeners at 41 diverse receptors, channels and a transporter. The step-wise truncation of rigidified, trisubstituted (at N6, C2, and 5′ positions) nucleosides revealed unanticipated interactions mainly with biogenic amine receptors, such as adrenergic receptors and serotonergic receptors, with affinities as high as 61 nM. The unmasking of consistent sets of structure activity relationship (SAR) at novel sites suggested similarities between receptor families in molecular recognition. Extensive molecular modeling of the GPCRs affected suggested binding modes of the ligands that supported the patterns of SAR at individual receptors. In some cases, the ligand docking mode closely resembled AR binding and in other cases the ligand assumed different orientations. The recognition patterns for different GPCRs were clustered according to which substituent groups were tolerated and explained in light of the complementarity with the receptor binding site. Thus, some likely off-target interactions, a concern for secondary drug effects, can be predicted for analogues of this set of substructures, aiding the design of additional structural analogues that either eliminate or accentuate certain off-target activities. Moreover, similar analyses could be performed for unrelated structural families for other GPCRs. PMID:24859150

Structural probing of off-target G protein-coupled receptor activities within a series of adenosine/adenine congeners.

PubMed

Paoletta, Silvia; Tosh, Dilip K; Salvemini, Daniela; Jacobson, Kenneth A

2014-01-01

We studied patterns of off-target receptor interactions, mostly at G protein-coupled receptors (GPCRs) in the µM range, of nucleoside derivatives that are highly engineered for nM interaction with adenosine receptors (ARs). Because of the considerable interest of using AR ligands for treating diseases of the CNS, we used the Psychoactive Drug Screening Program (PDSP) for probing promiscuity of these adenosine/adenine congeners at 41 diverse receptors, channels and a transporter. The step-wise truncation of rigidified, trisubstituted (at N6, C2, and 5' positions) nucleosides revealed unanticipated interactions mainly with biogenic amine receptors, such as adrenergic receptors and serotonergic receptors, with affinities as high as 61 nM. The unmasking of consistent sets of structure activity relationship (SAR) at novel sites suggested similarities between receptor families in molecular recognition. Extensive molecular modeling of the GPCRs affected suggested binding modes of the ligands that supported the patterns of SAR at individual receptors. In some cases, the ligand docking mode closely resembled AR binding and in other cases the ligand assumed different orientations. The recognition patterns for different GPCRs were clustered according to which substituent groups were tolerated and explained in light of the complementarity with the receptor binding site. Thus, some likely off-target interactions, a concern for secondary drug effects, can be predicted for analogues of this set of substructures, aiding the design of additional structural analogues that either eliminate or accentuate certain off-target activities. Moreover, similar analyses could be performed for unrelated structural families for other GPCRs.

Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

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

Bhaumik, Prasenjit; Xiao, Huogen; Parr, Charity L.

The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5-, 3.3-, and 3.05-{angstrom} resolutions, respectively. In the apoenzyme crystals, HAP forms a tight dimer not seen previously in any aspartic protease. The interactions between the monomers affect the conformation of two flexible loops, the functionally important 'flap' (residues 70-83) and its structural equivalent in the C-terminal domain (residues 238-245), as well as the orientation of helix 225-235. The flap is found in an open conformation in the apoenzyme. Unexpectedly, the active sitemore » of the apoenzyme contains a zinc ion tightly bound to His32 and Asp215 from one monomer and to Glu278A from the other monomer, with the coordination of Zn resembling that seen in metalloproteases. The flap is closed in the structure of the pepstatin A complex, whereas it is open in the complex with KNI-10006. Although the binding mode of pepstatin A is significantly different from that in other pepsin-like aspartic proteases, its location in the active site makes unlikely the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI-10006 is unusual compared with the binding of other inhibitors from the KNI series to aspartic proteases. The novel features of the HAP active site could facilitate design of specific inhibitors used in the development of antimalarial drugs.« less

Human La binds mRNAs through contacts to the poly(A) tail.

PubMed

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-05-04

In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.

Crystal structure of the S187F variant of human liver alanine: Aminotransferase associated with primary hyperoxaluria type I and its functional implications

PubMed Central

Oppici, Elisa; Fodor, Krisztian; Paiardini, Alessandro; Williams, Chris; Voltattorni, Carla Borri; Wilmanns, Matthias; Cellini, Barbara

2013-01-01

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5′-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results. Proteins 2013; 81:1457–1465. © 2013 Wiley Periodicals, Inc. PMID:23589421

Dynamic New World: Refining Our View of Protein Structure, Function and Evolution

PubMed Central

Mannige, Ranjan V.

2014-01-01

Proteins are crucial to the functioning of all lifeforms. Traditional understanding posits that a single protein occupies a single structure (“fold”), which performs a single function. This view is radically challenged with the recognition that high structural dynamism—the capacity to be extra “floppy”—is more prevalent in functional proteins than previously assumed. As reviewed here, this dynamic take on proteins affects our understanding of protein “structure”, function, and evolution, and even gives us a glimpse into protein origination. Specifically, this review will discuss historical developments concerning protein structure, and important new relationships between dynamism and aspects of protein sequence, structure, binding modes, binding promiscuity, evolvability, and origination. Along the way, suggestions will be provided for how key parts of textbook definitions—that so far have excluded membership to intrinsically disordered proteins (IDPs)—could be modified to accommodate our more dynamic understanding of proteins. PMID:28250374

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

PubMed Central

Kurkcuoglu, Zeynep; Bakan, Ahmet; Kocaman, Duygu; Bahar, Ivet; Doruker, Pemra

2012-01-01

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site. PMID:23028297

Solution NMR Analyses of the C-type Carbohydrate Recognition Domain of DC-SIGNR Protein Reveal Different Binding Modes for HIV-derived Oligosaccharides and Smaller Glycan Fragments

PubMed Central

Probert, Fay; Whittaker, Sara B.-M.; Crispin, Max; Mitchell, Daniel A.; Dixon, Ann M.

2013-01-01

The C-type lectin DC-SIGNR (dendritic cell-specific ICAM-3-grabbing non-integrin-related; also known as L-SIGN or CD299) is a promising drug target due to its ability to promote infection and/or within-host survival of several dangerous pathogens (e.g. HIV and severe acute respiratory syndrome coronavirus (SARS)) via interactions with their surface glycans. Crystallography has provided excellent insight into the mechanism by which DC-SIGNR interacts with small glycans, such as (GlcNAc)2Man3; however, direct observation of complexes with larger, physiological oligosaccharides, such as Man9GlcNAc2, remains elusive. We have utilized solution-state nuclear magnetic resonance spectroscopy to investigate DC-SIGNR binding and herein report the first backbone assignment of its active, calcium-bound carbohydrate recognition domain. Direct interactions with the small sugar fragments Man3, Man5, and (GlcNAc)2Man3 were investigated alongside Man9GlcNAc derived from recombinant gp120 (present on the HIV viral envelope), providing the first structural data for DC-SIGNR in complex with a virus-associated ligand, and unique binding modes were observed for each glycan. In particular, our data show that DC-SIGNR has a different binding mode for glycans on the HIV viral envelope compared with the smaller glycans previously observed in the crystalline state. This suggests that using the binding mode of Man9GlcNAc, instead of those of small glycans, may provide a platform for the design of DC-SIGNR inhibitors selective for high mannose glycans (like those on HIV). 15N relaxation measurements provided the first information on the dynamics of the carbohydrate recognition domain, demonstrating that it is a highly flexible domain that undergoes ligand-induced conformational and dynamic changes that may explain the ability of DC-SIGNR to accommodate a range of glycans on viral surfaces. PMID:23788638

Discovery and X-ray crystallographic analysis of a spiropiperidine iminohydantoin inhibitor of beta-secretase.

PubMed

Barrow, James C; Stauffer, Shaun R; Rittle, Kenneth E; Ngo, Phung L; Yang, ZhiQiang; Selnick, Harold G; Graham, Samuel L; Munshi, Sanjeev; McGaughey, Georgia B; Holloway, M Katharine; Simon, Adam J; Price, Eric A; Sankaranarayanan, Sethu; Colussi, Dennis; Tugusheva, Katherine; Lai, Ming-Tain; Espeseth, Amy S; Xu, Min; Huang, Qian; Wolfe, Abigail; Pietrak, Beth; Zuck, Paul; Levorse, Dorothy A; Hazuda, Daria; Vacca, Joseph P

2008-10-23

A high-throughput screen at 100 microM inhibitor concentration for the BACE-1 enzyme revealed a novel spiropiperidine iminohydantoin aspartyl protease inhibitor template. An X-ray cocrystal structure with BACE-1 revealed a novel mode of binding whereby the inhibitor interacts with the catalytic aspartates via bridging water molecules. Using the crystal structure as a guide, potent compounds with good brain penetration were designed.

Crystallization of Mitochondrial Respiratory Complex II from Chicken Heart: a Membrane Protein Complex Diffracting to 2.0 Å.

PubMed Central

Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward

2006-01-01

Synopsis A multi-subunit mitochondrial membrane protein complex involved in the Krebs Cycle and respiratory chain has been crystallized in a form suitable for near-atomic resolution structure determination. A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Å with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites. PMID:15805592

Interaction of phenazinium dyes with double-stranded poly(A): Spectroscopy and isothermal titration calorimetry studies

NASA Astrophysics Data System (ADS)

Khan, Asma Yasmeen; Saha, Baishakhi; Kumar, Gopinatha Suresh

2014-10-01

A comprehensive study on the binding of phenazinium dyes viz. janus green B, indoine blue, safranine O and phenosafranine with double stranded poly(A) using various spectroscopic and calorimetric techniques is presented. A higher binding of janus green B and indoine blue over safranine O and phenosafranine to poly(A) was observed from all experiments. Intercalative mode of binding of the dyes was inferred from fluorescence polarization anisotropy, iodide quenching and viscosity experiments. Circular dichroism study revealed significant perturbation of the secondary structure of poly(A) on binding of these dyes. Results from isothermal titration calorimetry experiments suggested that the binding was predominantly entropy driven with a minor contribution of enthalpy to the standard molar Gibbs energy. The results presented here may open new opportunities in the application of these dyes as RNA targeted therapeutic agents.

Structures of the tRNA export factor in the nuclear and cytosolic states.

PubMed

Cook, Atlanta G; Fukuhara, Noemi; Jinek, Martin; Conti, Elena

2009-09-03

Transfer RNAs are among the most ubiquitous molecules in cells, central to decoding information from messenger RNAs on translating ribosomes. In eukaryotic cells, tRNAs are actively transported from their site of synthesis in the nucleus to their site of function in the cytosol. This is mediated by a dedicated nucleo-cytoplasmic transport factor of the karyopherin-beta family (Xpot, also known as Los1 in Saccharomyces cerevisiae). Here we report the 3.2 A resolution structure of Schizosaccharomyces pombe Xpot in complex with tRNA and RanGTP, and the 3.1 A structure of unbound Xpot, revealing both nuclear and cytosolic snapshots of this transport factor. Xpot undergoes a large conformational change on binding cargo, wrapping around the tRNA and, in particular, binding to the tRNA 5' and 3' ends. The binding mode explains how Xpot can recognize all mature tRNAs in the cell and yet distinguish them from those that have not been properly processed, thus coupling tRNA export to quality control.

Insight into the binding modes of Lassa nucleoprotein complexed with ssRNA by molecular dynamic simulations and free energy calculations.

PubMed

Zhang, Ying; Chen, Hang; Han, Ju-Guang

2015-01-01

Lassa virus (LASV), an arenavirus known to be responsible for a severe hemorrhagic fever, causes thousands of deaths annually and there is no effective vaccine for it so far. The nucleoprotein (NP) of LASV plays an essential role in the replication and transcription of the viral genome. Recent research shows that viral RNA binds in a deep crevice located within the N-terminal domain of NP and suggests a gating mechanism in which NP transforms from a "closed" position to an "open" position to bind RNA. To characterize the molecular mechanisms of how RNA binds to N-terminal domain of NP, two molecular dynamic (MD) simulations of RNA-binding structure and RNA-free structure have been performed. The simulation results show that an important helix α6 interacts with RNA in the "open" conformation, while helix α6 rotates toward the binding crevice and reduces the space of RNA-binding pocket in the "closed" conformation; it appears that helix α6 would clash with RNA while NP is in a "closed" state. In addition, to characterize the role of residues involved in the binding of RNA, the MD simulations of the double-mutant (W164A/F176A) and the single-mutant (G243P) RNA-binding NP complexes have been performed. Our MD simulations and molecular mechanics-generalized born surface area (MM-GBSA) energy calculations exhibit that the three mutant residues increase the binding affinity. Furthermore, we infer that the defect of the replication and transcription of viral genome is possibly due to the change of structural integrity rather than the reduction of RNA-binding affinity.

Structural Basis for Activation of Fatty Acid-binding Protein 4

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

Gillilan,R.; Ayers, S.; Noy, N.

2007-01-01

Fatty acid-binding protein 4 (FABP4) delivers ligands from the cytosol to the nuclear receptor PPAR{gamma} in the nucleus, thereby enhancing the transcriptional activity of the receptor. Notably, FABP4 binds multiple ligands with a similar affinity but its nuclear translocation is activated only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of the nuclear import of FABP4, we solved the crystal structures of the protein complexed with two compounds that induce its nuclear translocation, and compared these to the apo-protein and to FABP4 structures bound to non-activating ligands. Examination of these structures indicatesmore » that activation coincides with closure of a portal loop phenylalanine side-chain, contraction of the binding pocket, a subtle shift in a helical domain containing the nuclear localization signal of the protein, and a resultant change in oligomeric state that exposes the nuclear localization signal to the solution. Comparisons of backbone displacements induced by activating ligands with a measure of mobility derived from translation, libration, screw (TLS) refinement, and with a composite of slowest normal modes of the apo state suggest that the helical motion associated with the activation of the protein is part of the repertoire of the equilibrium motions of the apo-protein, i.e. that ligand binding does not induce the activated configuration but serves to stabilize it. Nuclear import of FABP4 can thus be understood in terms of the pre-existing equilibrium hypothesis of ligand binding.« less

Structural and functional insight into the carbohydrate receptor binding of F4 fimbriae-producing enterotoxigenic Escherichia coli.

PubMed

Moonens, Kristof; Van den Broeck, Imke; De Kerpel, Maia; Deboeck, Francine; Raymaekers, Hanne; Remaut, Han; De Greve, Henri

2015-03-27

Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4ab, F4ac, and F4ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeGad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D'-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe(150)-Glu(152) and Val(166)-Glu(170) of FaeGad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D'-α1 loop that borders the FaeGad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular docking.

PubMed

Morris, Garrett M; Lim-Wilby, Marguerita

2008-01-01

Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. The goal of ligand-protein docking is to predict the predominant binding mode(s) of a ligand with a protein of known three-dimensional structure. Successful docking methods search high-dimensional spaces effectively and use a scoring function that correctly ranks candidate dockings. Docking can be used to perform virtual screening on large libraries of compounds, rank the results, and propose structural hypotheses of how the ligands inhibit the target, which is invaluable in lead optimization. The setting up of the input structures for the docking is just as important as the docking itself, and analyzing the results of stochastic search methods can sometimes be unclear. This chapter discusses the background and theory of molecular docking software, and covers the usage of some of the most-cited docking software.

Fast and automated functional classification with MED-SuMo: an application on purine-binding proteins.

PubMed

Doppelt-Azeroual, Olivia; Delfaud, François; Moriaud, Fabrice; de Brevern, Alexandre G

2010-04-01

Ligand-protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects.

Fast and automated functional classification with MED-SuMo: An application on purine-binding proteins

PubMed Central

Doppelt-Azeroual, Olivia; Delfaud, François; Moriaud, Fabrice; de Brevern, Alexandre G

2010-01-01

Ligand–protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects. PMID:20162627

Molecular dynamics studies of the 3D structure and planar ligand binding of a quadruplex dimer.

PubMed

Li, Ming-Hui; Luo, Quan; Xue, Xiang-Gui; Li, Ze-Sheng

2011-03-01

G-rich sequences can fold into a four-stranded structure called a G-quadruplex, and sequences with short loops are able to aggregate to form stable quadruplex multimers. Few studies have characterized the properties of this variety of quadruplex multimers. Using molecular modeling and molecular dynamics simulations, the present study investigated a dimeric G-quadruplex structure formed from a simple sequence of d(GGGTGGGTGGGTGGGT) (G1), and its interactions with a planar ligand of a perylene derivative (Tel03). A series of analytical methods, including free energy calculations and principal components analysis (PCA), was used. The results show that a dimer structure with stacked parallel monomer structures is maintained well during the entire simulation. Tel03 can bind to the dimer efficiently through end stacking, and the binding mode of the ligand stacked with the 3'-terminal thymine base is most favorable. PCA showed that the dominant motions in the free dimer occur on the loop regions, and the presence of the ligand reduces the flexibility of the loops. Our investigation will assist in understanding the geometric structure of stacked G-quadruplex multimers and may be helpful as a platform for rational drug design.

Spectroscopic investigation of new water soluble Mn(II)(2) and Mg(II)(2) complexes for the substrate binding models of xylose/glucose isomerases.

PubMed

Patra, Ayan; Bera, Manindranath

2014-01-30

In methanol, the reaction of stoichiometric amounts of Mn(OAc)(2)·4H(2)O and the ligand H(3)hpnbpda [H(3)hpnbpda=N,N'-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N'-diacetic acid] in the presence of NaOH, afforded a new water soluble dinuclear manganese(II) complex, [Mn2(hpnbpda)(μ-OAc)] (1). Similarly, the reaction of Mg(OAc)(2)·4H(2)O and the ligand H3hpnbpda in the presence of NaOH, in methanol, yielded a new water soluble dinuclear magnesium(II) complex, [Mg2(hpnbpda)(μ-OAc)(H2O)2] (2). DFT calculations have been performed for the structural optimization of complexes 1 and 2. The DFT optimized structure of complex 1 shows that two manganese(II) centers are in a distorted square pyramidal geometry, whereas the DFT optimized structure of complex 2 reveals that two magnesium(II) centers adopt a six-coordinate distorted octahedral geometry. To understand the mode of substrate binding and the mechanistic details of the active site metals in xylose/glucose isomerases (XGI), we have investigated the binding interactions of biologically important monosaccharides d-glucose and d-xylose with complexes 1 and 2, in aqueous alkaline solution by a combined approach of FTIR, UV-vis, fluorescence, and (13)C NMR spectroscopic techniques. Fluorescence spectra show the binding-induced gradual decrease in emission of complexes 1 and 2 accompanied by a significant blue shift upon increasing the concentration of sugar substrates. The binding modes of d-glucose and d-xylose with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in (13)C NMR spectra for C1 and C2 carbon atoms. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pub1p C-Terminal RRM Domain Interacts with Tif4631p through a Conserved Region Neighbouring the Pab1p Binding Site

PubMed Central

Rico-Lastres, Palma; Pérez-Cañadillas, José Manuel

2011-01-01

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1–402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role. PMID:21931728

pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families.

PubMed

Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Svedas, Vytas

2014-07-01

The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure-function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular docking and dynamics simulation study on the influence of Zn2+ on the binding modes of aggrecanase with its inhibitors.

PubMed

Suganya, Panneer S R; Kalva, Sukesh; Saleena, Lilly M

2014-01-01

Zinc plays a vital role in structural organization, regulation of function and stabilization of the folded protein, which ultimately activates or inactivates the binding sites of the protein. Its transition makes a major change in the protein and its binding affinity. The ligand binding aggrecanases can be influenced by Zn2+ ions; therefore the study focuses on checking the binding mode in the presence and absence of zinc using Docking and Molecular dynamics simulation. The crystal structure with zinc was considered as wild type (ADAMTS-4-1Zn2+, ADAMTS-5-1Zn2+) and the crystal structure without zinc was considered as the mutant type (ADAMTS-4-0Zn2+, ADAMTS-5-0Zn2+). Mutations were made manually by deleting the zinc atom. ADAMTS-4-1Zn2+ had the best Glide score of -12.66 kcal·mol−1, whereas ADAMTS-4-0Zn2+ had -11.69 kcal·mol−1. ADAMTS-4-1Zn2+ had the best glide energy of -72.29 kcal·mol−1, whereas ADAMTS-4-0Zn2+ had-68.44 kcal·mol−1. ADAMTS-4-1Zn2+ had the best glide e-model of -116.34, whereas ADAMTS-4-0Zn2+ had -104.264. The RMSD value for ADAMTS-4-1Zn2+ and ADAMTS-4-0Zn2+ was 1.9. These results suggested that the absence of zinc decreases the binding affinity of ADAMTS-4 with its inhibitor. ADAMTS-5-1Zn2+ had the best Glide score of -8.32 kcal·mol−1, whereas ADAMTS-5-0Zn2+ had -6.62 kcal·mol−1. ADAMTS-5-1Zn2+ had the best glide energy of -70.28 kcal·mol−1, whereas ADAMTS-5-0Zn2+ had -66.02 kcal·mol−1. ADAMTS-5-1Zn2+ had the best glide e-model of-108.484, whereas ADAMTS-5-0Zn2+ had -93.81. The RMSD value for ADAMTS-5-1Zn2+ and ADAMTS-5-0Zn2+ was 0.48Å. These results confirmed that the absence of zinc decreased the binding affinity of ADAMTS-5 with its inhibitor whereas the presence extended the docking energy range and strengthened the binding affinity. Per-residue interaction study, MM-GBSA and Molecular Dynamics showed that all the four complexes underwent extensive structural changes whereas the complex with zinc was stable throughout the simulation period.

Motion of spin label side chains in cellular retinol-binding protein: correlation with structure and nearest-neighbor interactions in an antiparallel beta-sheet.

PubMed

Lietzow, Michael A; Hubbell, Wayne L

2004-03-23

A goal in the development of site-directed spin labeling in proteins is to correlate the motion of a nitroxide side chain with local structure, interactions, and dynamics. Significant progress toward this goal has been made using alpha-helical proteins of known structure, and the present study is the first step in a similar exploration of a beta-sheet protein, cellular retinol-binding protein (CRBP). Nitroxide side chains were introduced along both interior and edge strands. At sites in interior strands, the side-chain motion is strongly influenced by interactions with side chains of neighboring strands, giving rise to a rich variety of dynamic modes (weakly ordered, strongly ordered, immobilized) and complex electron paramagnetic resonance spectra that are modulated by strand twist. The interactions giving rise to the dynamic modes are explored using mutagenesis, and the results demonstrate the particular importance of the non-hydrogen-bonded neighbor residue in giving rise to highly ordered states. Along edge strands of the beta-sheet, the motion of the side chain is simple and weakly ordered, resembling that at solvent-exposed surfaces of an alpha-helix. A simple working model is proposed that can account for the wide variety of dynamic modes encountered. Collectively, the results suggest that the nitroxide side chain is an effective probe of side-chain interactions, and that site-directed spin labeling should be a powerful means of monitoring conformational changes that involve changes in beta-sheet topology.

Crystal Structure of the Cohesin Gatekeeper Pds5 and in Complex with Kleisin Scc1.

PubMed

Lee, Byung-Gil; Roig, Maurici B; Jansma, Marijke; Petela, Naomi; Metson, Jean; Nasmyth, Kim; Löwe, Jan

2016-03-08

Sister chromatid cohesion is mediated by cohesin, whose Smc1, Smc3, and kleisin (Scc1) subunits form a ring structure that entraps sister DNAs. The ring is opened either by separase, which cleaves Scc1 during anaphase, or by a releasing activity involving Wapl, Scc3, and Pds5, which bind to Scc1 and open its interface with Smc3. We present crystal structures of Pds5 from the yeast L. thermotolerans in the presence and absence of the conserved Scc1 region that interacts with Pds5. Scc1 binds along the spine of the Pds5 HEAT repeat fold and is wedged between the spine and C-terminal hook of Pds5. We have isolated mutants that confirm the observed binding mode of Scc1 and verified their effect on cohesin by immunoprecipitation and calibrated ChIP-seq. The Pds5 structure also reveals architectural similarities to Scc3, the other large HEAT repeat protein of cohesin and, most likely, Scc2. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

An exclusive α/β code directs allostery in TetR-peptide complexes.

PubMed

Sevvana, Madhumati; Goetz, Christoph; Goeke, Dagmar; Wimmer, Cornelius; Berens, Christian; Hillen, Wolfgang; Muller, Yves A

2012-02-10

The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure-function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended β-like conformation, two anti-inducing peptides form an α-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular Structures and Functional Relationships in Clostridial Neurotoxins

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

Swaminathan S.

2011-12-01

The seven serotypes of Clostridium botulinum neurotoxins (A-G) are the deadliest poison known to humans. They share significant sequence homology and hence possess similar structure-function relationships. Botulinum neurotoxins (BoNT) act via a four-step mechanism, viz., binding and internalization to neuronal cells, translocation of the catalytic domain into the cytosol and finally cleavage of one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) causing blockage of neurotransmitter release leading to flaccid paralysis. Crystal structures of three holotoxins, BoNT/A, B and E, are available to date. Although the individual domains are remarkably similar, their domain organization is different. These structuresmore » have helped in correlating the structural and functional domains. This has led to the determination of structures of individual domains and combinations of them. Crystal structures of catalytic domains of all serotypes and several binding domains are now available. The catalytic domains are zinc endopeptidases and share significant sequence and structural homology. The active site architecture and the catalytic mechanism are similar although the binding mode of individual substrates may be different, dictating substrate specificity and peptide cleavage selectivity. Crystal structures of catalytic domains with substrate peptides provide clues to specificity and selectivity unique to BoNTs. Crystal structures of the receptor domain in complex with ganglioside or the protein receptor have provided information about the binding of botulinum neurotoxin to the neuronal cell. An overview of the structure-function relationship correlating the 3D structures with biochemical and biophysical data and how they can be used for structure-based drug discovery is presented here.« less

Structure of the Varicella Zoster Virus Thymidylate Synthase Establishes Functional and Structural Similarities as the Human Enzyme and Potentiates Itself as a Target of Brivudine

PubMed Central

Hew, Kelly; Dahlroth, Sue-Li; Veerappan, Saranya; Pan, Lucy Xin; Cornvik, Tobias; Nordlund, Pär

2015-01-01

Varicella zoster virus (VZV) is a highly infectious human herpesvirus that is the causative agent for chicken pox and shingles. VZV encodes a functional thymidylate synthase (TS), which is the sole enzyme that produces dTMP from dUMP de novo. To study substrate binding, the complex structure of TSVZV with dUMP was determined to a resolution of 2.9 Å. In the absence of a folate co-substrate, dUMP binds in the conserved TS active site and is coordinated similarly as in the human encoded TS (TSHS) in an open conformation. The interactions between TSVZV with dUMP and a cofactor analog, raltitrexed, were also studied using differential scanning fluorimetry (DSF), suggesting that TSVZV binds dUMP and raltitrexed in a sequential binding mode like other TS. The DSF also revealed interactions between TSVZV and in vitro phosphorylated brivudine (BVDUP), a highly potent anti-herpesvirus drug against VZV infections. The binding of BVDUP to TSVZV was further confirmed by the complex structure of TSVZV and BVDUP solved at a resolution of 2.9 Å. BVDUP binds similarly as dUMP in the TSHS but it induces a closed conformation of the active site. The structure supports that the 5-bromovinyl substituent on BVDUP is likely to inhibit TSVZV by preventing the transfer of a methylene group from its cofactor and the subsequent formation of dTMP. The interactions between TSVZV and BVDUP are consistent with that TSVZV is indeed a target of brivudine in vivo. The work also provided the structural basis for rational design of more specific TSVZV inhibitors. PMID:26630264

Structure of a Yeast Dyn2-Nup159 Complex and Molecular Basis for Dynein Light Chain-Nuclear Pore Interaction*

PubMed Central

Romes, Erin M.; Tripathy, Ashutosh; Slep, Kevin C.

2012-01-01

The nuclear pore complex gates nucleocytoplasmic transport through a massive, eight-fold symmetric channel capped by a nucleoplasmic basket and structurally unique, cytoplasmic fibrils whose tentacles bind and regulate asymmetric traffic. The conserved Nup82 complex, composed of Nsp1, Nup82, and Nup159, forms the unique cytoplasmic fibrils that regulate mRNA nuclear export. Although the nuclear pore complex plays a fundamental, conserved role in nuclear trafficking, structural information about the cytoplasmic fibrils is limited. Here, we investigate the structural and biochemical interactions between Saccharomyces cerevisiae Nup159 and the nucleoporin, Dyn2. We find that Dyn2 is predominantly a homodimer and binds arrayed sites on Nup159, promoting the Nup159 parallel homodimerization. We present the first structure of Dyn2, determined at 1.85 Å resolution, complexed with a Nup159 target peptide. Dyn2 resembles homologous metazoan dynein light chains, forming homodimeric composite substrate binding sites that engage two independent 10-residue target motifs, imparting a β-strand structure to each peptide via antiparallel extension of the Dyn2 core β-sandwich. Dyn2 recognizes a highly conserved QT motif while allowing sequence plasticity in the flanking residues of the peptide. Isothermal titration calorimetric analysis of the comparative binding of Dyn2 to two Nup159 target sites shows similar affinities (18 and 13 μm), but divergent thermal binding modes. Dyn2 homodimers are arrayed in the crystal lattice, likely mimicking the arrayed architecture of Dyn2 on the Nup159 multivalent binding sites. Crystallographic interdimer interactions potentially reflect a cooperative basis for Dyn2-Nup159 complex formation. Our data highlight the determinants that mediate oligomerization of the Nup82 complex and promote a directed, elongated cytoplasmic fibril architecture. PMID:22411995

Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment

PubMed Central

Mollica, Luca; Bessa, Luiza M.; Hanoulle, Xavier; Jensen, Malene Ringkjøbing; Blackledge, Martin; Schneider, Robert

2016-01-01

In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the “fly-casting” hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context. PMID:27668217

Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA

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

Xing, G.; Kirouac, K.; Shin, Y.J.

2009-09-16

DNA polymerases are co-ordinated by sliding clamps (PCNA/{beta}-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1-PCNA2 at 2.05 {angstrom} resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with amore » 3{sub 10} helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein-protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.« less

A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms

NASA Astrophysics Data System (ADS)

Jones, Alan M.; Westwood, Isaac M.; Osborne, James D.; Matthews, Thomas P.; Cheeseman, Matthew D.; Rowlands, Martin G.; Jeganathan, Fiona; Burke, Rosemary; Lee, Diane; Kadi, Nadia; Liu, Manjuan; Richards, Meirion; McAndrew, Craig; Yahya, Norhakim; Dobson, Sarah E.; Jones, Keith; Workman, Paul; Collins, Ian; van Montfort, Rob L. M.

2016-10-01

The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops.

In vitro and in silico assessment of the structure-dependent binding of bisphenol analogues to glucocorticoid receptor.

PubMed

Zhang, Jie; Zhang, Tiehua; Guan, Tianzhu; Yu, Hansong; Li, Tiezhu

2017-03-01

Widespread use of bisphenol A (BPA) and other bisphenol analogues has attracted increasing attention for their potential adverse effects. As environmental endocrine-disrupting compounds (EDCs), bisphenols (BPs) may activate a variety of nuclear receptors, including glucocorticoid receptor (GR). In this work, the binding of 11 BPs to GR was investigated by fluorescence polarization (FP) assay in combination with molecular dynamics simulations. The human glucocorticoid receptor was prepared as a soluble recombinant protein. A fluorescein-labeled dexamethasone derivative (Dex-fl) was employed as tracer. Competitive displacement of Dex-fl from GR by BPs showed that the binding affinities of bisphenol analogues were largely dependent on their characteristic functional groups. In order to further understand the relationship between BPs structures and their GR-mediated activities, molecular docking was utilized to explore the binding modes at the atomic level. The results confirmed that structural variations of bisphenol analogues contributed to different interactions of BPs with GR, potentially causing distinct toxic effects. Comparison of the calculated binding energies vs. experimental binding affinities yielded a good correlation (R 2  = 0.8266), which might be helpful for the design of environmentally benign materials with reduced toxicities. In addition, the established FP assay based on GR exhibited the potential to offer an alternative to traditional methods for the detection of bisphenols.

pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families

PubMed Central

Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Švedas, Vytas

2014-01-01

The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure–function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. PMID:24852248

Alternative binding modes identified for growth and differentiation factor-associated serum protein (GASP) family antagonism of myostatin.

PubMed

Walker, Ryan G; Angerman, Elizabeth B; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B

2015-03-20

Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Alternative Binding Modes Identified for Growth and Differentiation Factor-associated Serum Protein (GASP) Family Antagonism of Myostatin*

PubMed Central

Walker, Ryan G.; Angerman, Elizabeth B.; Kattamuri, Chandramohan; Lee, Yun-Sil; Lee, Se-Jin; Thompson, Thomas B.

2015-01-01

Myostatin, a member of the TGF-β family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-β family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-β antagonism, where closely related antagonists can utilize different ligand-binding strategies. PMID:25657005

Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

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

Sprenger, Janina; Lund University, SE-221 84 Lund; Svensson, Bo

In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substratemore » putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.« less

Abnormal swelling of the peritrophic membrane in Eri silkworm gut caused by MLX56 family defense proteins with chitin-binding and extensin domains.

PubMed

Konno, Kotaro; Shimura, Sachiko; Ueno, Chihiro; Arakawa, Toru; Nakamura, Masatoshi

2018-03-01

MLX56 family defense proteins, MLX56 and its close homolog LA-b, are chitin-binding defense proteins found in mulberry latex that show strong growth-inhibitions against caterpillars when fed at concentrations as low as 0.01%. MLX56 family proteins contain a unique structure with an extensin domain surrounded by two hevein-like chitin-binding domains, but their defensive modes of action remain unclear. Here, we analyzed the effects of MLX56 family proteins on the peritrophic membrane (PM), a thin and soft membrane consisting of chitin that lines the midgut lumen of insects. We observed an abnormally thick (>1/5 the diameter of midgut) hard gel-like membrane consisted of chitin and MLX56 family proteins, MLX56 and LA-b, in the midgut of the Eri silkworms, Samia ricini, fed a diet containing MLX56 family proteins, MLX56 and LA-b. When polyoxin AL, a chitin-synthesis-inhibitor, was added to the diet containing MLX56 family proteins, the toxicity of MLX56 family proteins disappeared and PM became thinner and fragmented. These results suggest that MLX56 family proteins, through their chitin-binding domains, bind to the chitin framework of PM, then through their extensin-domain (gum arabic-like structure), which functions as swelling agent, expands PM into an abnormally thick membrane that inhibits the growth of insects. This study shows that MLX56 family proteins are plant defense lectins with a totally unique mode of action, and reveals the functions of extensin domains and arabinogalactan proteins as swelling (gel-forming) agents of plants. Copyright © 2018 Elsevier Ltd. All rights reserved.

New Method to Study the Vibrational Modes of Biomolecules in the Terahertz Range Based on a Single-Stage Raman Spectrometer.

PubMed

Kalanoor, Basanth S; Ronen, Maria; Oren, Ziv; Gerber, Doron; Tischler, Yaakov R

2017-03-31

The low-frequency vibrational (LFV) modes of biomolecules reflect specific intramolecular and intermolecular thermally induced fluctuations that are driven by external perturbations, such as ligand binding, protein interaction, electron transfer, and enzymatic activity. Large efforts have been invested over the years to develop methods to access the LFV modes due to their importance in the studies of the mechanisms and biological functions of biomolecules. Here, we present a method to measure the LFV modes of biomolecules based on Raman spectroscopy that combines volume holographic filters with a single-stage spectrometer, to obtain high signal-to-noise-ratio spectra in short acquisition times. We show that this method enables LFV mode characterization of biomolecules even in a hydrated environment. The measured spectra exhibit distinct features originating from intra- and/or intermolecular collective motion and lattice modes. The observed modes are highly sensitive to the overall structure, size, long-range order, and configuration of the molecules, as well as to their environment. Thus, the LFV Raman spectrum acts as a fingerprint of the molecular structure and conformational state of a biomolecule. The comprehensive method we present here is widely applicable, thus enabling high-throughput study of LFV modes of biomolecules.

Detection and characterization of nonspecific, sparsely-populated binding modes in the early stages of complexation

PubMed Central

Cardone, A.; Bornstein, A.; Pant, H. C.; Brady, M.; Sriram, R.; Hassan, S. A.

2015-01-01

A method is proposed to study protein-ligand binding in a system governed by specific and non-specific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultra-weak associations lead instead to broader distributions, a manifestation of non-specific, sparsely-populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first-encounter modes are metastable states from which stable (pre-relaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide-protein complex. The peptide adopts several low-population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated. PMID:25782918

Structural, spectral, DFT and biological studies on macrocyclic mononuclear ruthenium (II) complexes

NASA Astrophysics Data System (ADS)

Muthukkumar, M.; Kamal, C.; Venkatesh, G.; Kaya, C.; Kaya, S.; Enoch, Israel V. M. V.; Vennila, P.; Rajavel, R.

2017-11-01

Macrocyclic mononuclear ruthenium (II) complexes have been synthesized by condensation method [Ru (L1, L2, L3) Cl2] L1 = (C36 H31 N9), L2= (C42H36N8), L3= (C32H32 N8)]. These ruthenium complexes have been established by elemental analyses and spectroscopic techniques (Fourier transform infrared spectroscopy (FT-IR), 1H- nuclear magnetic resonance (NMR), 13C- NMR and Electrospray ionization mass spectrometry (ESI-MS)). The coordination mode of the ligand has been confirmed and the octahedral geometry around the ruthenium ion has been revealed. Binding affinity and binding mode of ruthenium (II) complexes with Bovine serum Albumin (BSA) have been characterized by Emission spectra analysis. UV-Visible and fluorescence spectroscopic techniques have also been utilized to examine the interaction between ligand and its complexes L1, L2, & L3 with BSA. Chemical parameters and molecular structure of Ru (II) complexes L1H, L2H, & L3H have been determined by DFT coupled with B3LYP/6-311G** functional in both the gaseous and aqueous phases.

Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1: 2,5-dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators.

PubMed

Han, Changho; Chatterjee, Arindam; Noetzel, Meredith J; Panarese, Joseph D; Smith, Emery; Chase, Peter; Hodder, Peter; Niswender, Colleen; Conn, P Jeffrey; Lindsley, Craig W; Stauffer, Shaun R

2015-01-15

Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure. Copyright © 2014 Elsevier Ltd. All rights reserved.

Peptidic β-sheet binding with Congo Red allows both reduction of error variance and signal amplification for immunoassays.

PubMed

Wang, Yunyun; Liu, Ye; Deng, Xinli; Cong, Yulong; Jiang, Xingyu

2016-12-15

Although conventional enzyme-linked immunosorbent assays (ELISA) and related assays have been widely applied for the diagnosis of diseases, many of them suffer from large error variance for monitoring the concentration of targets over time, and insufficient limit of detection (LOD) for assaying dilute targets. We herein report a readout mode of ELISA based on the binding between peptidic β-sheet structure and Congo Red. The formation of peptidic β-sheet structure is triggered by alkaline phosphatase (ALP). For the detection of P-Selectin which is a crucial indicator for evaluating thrombus diseases in clinic, the 'β-sheet and Congo Red' mode significantly decreases both the error variance and the LOD (from 9.7ng/ml to 1.1 ng/ml) of detection, compared with commercial ELISA (an existing gold-standard method for detecting P-Selectin in clinic). Considering the wide range of ALP-based antibodies for immunoassays, such novel method could be applicable to the analysis of many types of targets. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional map of arrestin binding to phosphorylated opsin, with and without agonist.

PubMed

Peterhans, Christian; Lally, Ciara C M; Ostermaier, Martin K; Sommer, Martha E; Standfuss, Jörg

2016-06-28

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct.

Interaction between Pin1 and its natural product inhibitor epigallocatechin-3-gallate by spectroscopy and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Xi, Lei; Wang, Yu; He, Qing; Zhang, Qingyan; Du, Linfang

2016-12-01

The binding of epigallocatechin-3-gallate (EGCG) to wild type Pin1 in solution was studied by spectroscopic methods and molecular dynamics simulations in this research to explore the binding mode and inhibition mechanism. The binding constants and number of binding sites per Pin1 for EGCG were calculated through the Stern-Volmer equation. The values of binding free energy and thermodynamic parameters were calculated and indicated that hydrogen bonds, electrostatic interaction and Van der Waals interaction played the major role in the binding process. The alterations of Pin1 secondary structure in the presence of EGCG were confirmed by far-UV circular dichroism spectra. The binding model at atomic-level revealed that EGCG was bound to the Glu12, Lys13, Arg14, Met15 and Arg17 in WW domain. Furthermore, EGCG could also interact with Arg69, Asp112, Cys113 and Ser114 in PPIase domain.

Human La binds mRNAs through contacts to the poly(A) tail

PubMed Central

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-01-01

Abstract In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3’OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3’OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail. PMID:29447394

Template-Based Modeling of Protein-RNA Interactions

PubMed Central

Zheng, Jinfang; Kundrotas, Petras J.; Vakser, Ilya A.

2016-01-01

Protein-RNA complexes formed by specific recognition between RNA and RNA-binding proteins play an important role in biological processes. More than a thousand of such proteins in human are curated and many novel RNA-binding proteins are to be discovered. Due to limitations of experimental approaches, computational techniques are needed for characterization of protein-RNA interactions. Although much progress has been made, adequate methodologies reliably providing atomic resolution structural details are still lacking. Although protein-RNA free docking approaches proved to be useful, in general, the template-based approaches provide higher quality of predictions. Templates are key to building a high quality model. Sequence/structure relationships were studied based on a representative set of binary protein-RNA complexes from PDB. Several approaches were tested for pairwise target/template alignment. The analysis revealed a transition point between random and correct binding modes. The results showed that structural alignment is better than sequence alignment in identifying good templates, suitable for generating protein-RNA complexes close to the native structure, and outperforms free docking, successfully predicting complexes where the free docking fails, including cases of significant conformational change upon binding. A template-based protein-RNA interaction modeling protocol PRIME was developed and benchmarked on a representative set of complexes. PMID:27662342

The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding.

PubMed

Prchal, Jan; Srb, Pavel; Hunter, Eric; Ruml, Tomáš; Hrabal, Richard

2012-10-26

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein. Copyright © 2012. Published by Elsevier Ltd.

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

PubMed

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

2016-02-04

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

Structural and Spectroscopic Analysis of the Kinase Inhibitor Bosutinib and an Isomer of Bosutinib Binding to the Abl Tyrosine Kinase Domain

PubMed Central

Levinson, Nicholas M.; Boxer, Steven G.

2012-01-01

Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl, which explains the inhibitor's activity against several imatinib-resistant mutants, and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name “bosutinib”, and report spectroscopic and structural characterizations of both. We show that the fluorescence properties of these compounds allow inhibitor binding to be measured quantitatively, and that the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases. Exploiting such differences could lead to inhibitors with improved selectivity. PMID:22493660

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling.

PubMed

Ajmal, Mohammad Rehan; Zaidi, Nida; Alam, Parvez; Nusrat, Saima; Siddiqi, Mohd Khursheed; Badr, Gamal; Mahmoud, Mohamed H; Khan, Rizwan Hasan

2017-01-01

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10 4  M -1 , and with the increase in temperature, Stern-Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV-visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug-albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R o ) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.

Mechanism for the Inhibition of the Carboxyl-transferase

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

L Yu; Y Kim; L Tong

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and have been targeted for drug development against obesity, diabetes, and other diseases. The carboxyltransferase (CT) domain of this enzyme is the site of action for three different classes of herbicides, as represented by haloxyfop, tepraloxydim, and pinoxaden. Our earlier studies have demonstrated that haloxyfop and tepraloxydim bind in the CT active site at the interface of its dimer. However, the two compounds probe distinct regions of the dimer interface, sharing primarily only two common anchoring points of interaction with the enzyme. We report here the crystal structure of the CT domain ofmore » yeast ACC in complex with pinoxaden at 2.8-{angstrom} resolution. Despite their chemical diversity, pinoxaden has a similar binding mode as tepraloxydim and requires a small conformational change in the dimer interface for binding. Crystal structures of the CT domain in complex with all three classes of herbicides confirm the importance of the two anchoring points for herbicide binding. The structures also provide a foundation for understanding the molecular basis of the herbicide resistance mutations and cross resistance among the herbicides, as well as for the design and development of new inhibitors against plant and human ACCs.« less

Electronic structure of antibiotic erythromycin

NASA Astrophysics Data System (ADS)

Novak, Igor; Kovač, Branka

2015-03-01

The electronic structure of erythromycin A (ERYMA) molecule has been studied by UV photoelectron spectroscopy and assigned (in the low ionization energy region only) by empirical arguments. The two orbitals with highest energy (lowest ionization energy) are localized on the nitrogen of the desosamine sugar functional group and on the ester group of macrolide (lactone) ring. We discuss how these orbital energies can help to rationalize the known mode of binding of ERYMA to their biological receptors.

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.

More powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules

PubMed Central

Spyrou, John A. B.; Kelly, James; Ren, Jingshan; Grimes, Jonathan; Puerstinger, Gerhard; Stonehouse, Nicola; Walter, Thomas S.; Hu, Zhongyu; Wang, Junzhi; Li, Xuemei; Peng, Wei; Rowlands, David; Fry, Elizabeth E.; Rao, Zihe; Stuart, David I.

2014-01-01

Enterovirus 71 (HEV71) epidemics amongst children and infants result mainly in mild symptoms, however, especially in the Asia-Pacific region, infection can be fatal. At present no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(-4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities, pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics-enhanced ligand docking, and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (IC50 = 25 pM) is an order of magnitude more potent than the best previously reported inhibitor, and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections. PMID:24509833

Structural virology. Near-atomic cryo-EM structure of the helical measles virus nucleocapsid.

PubMed

Gutsche, Irina; Desfosses, Ambroise; Effantin, Grégory; Ling, Wai Li; Haupt, Melina; Ruigrok, Rob W H; Sachse, Carsten; Schoehn, Guy

2015-05-08

Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus nucleocapsid offers important insights into the mechanism of the helical polymerization of nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design. Copyright © 2015, American Association for the Advancement of Science.

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA.

PubMed

Gantchev, Tsvetan G; Hunting, Darel J

2010-12-28

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.

Predicting bioactive conformations and binding modes of macrocycles

NASA Astrophysics Data System (ADS)

Anighoro, Andrew; de la Vega de León, Antonio; Bajorath, Jürgen

2016-10-01

Macrocyclic compounds experience increasing interest in drug discovery. It is often thought that these large and chemically complex molecules provide promising candidates to address difficult targets and interfere with protein-protein interactions. From a computational viewpoint, these molecules are difficult to treat. For example, flexible docking of macrocyclic compounds is hindered by the limited ability of current docking approaches to optimize conformations of extended ring systems for pose prediction. Herein, we report predictions of bioactive conformations of macrocycles using conformational search and binding modes using docking. Conformational ensembles generated using specialized search technique of about 70 % of the tested macrocycles contained accurate bioactive conformations. However, these conformations were difficult to identify on the basis of conformational energies. Moreover, docking calculations with limited ligand flexibility starting from individual low energy conformations rarely yielded highly accurate binding modes. In about 40 % of the test cases, binding modes were approximated with reasonable accuracy. However, when conformational ensembles were subjected to rigid body docking, an increase in meaningful binding mode predictions to more than 50 % of the test cases was observed. Electrostatic effects did not contribute to these predictions in a positive or negative manner. Rather, achieving shape complementarity at macrocycle-target interfaces was a decisive factor. In summary, a combined computational protocol using pre-computed conformational ensembles of macrocycles as a starting point for docking shows promise in modeling binding modes of macrocyclic compounds.

Mode of bindings of zinc oxide nanoparticles to myoglobin and horseradish peroxidase: A spectroscopic investigations

NASA Astrophysics Data System (ADS)

Mandal, Gopa; Bhattacharya, Sudeshna; Ganguly, Tapan

2011-07-01

The interactions between two heme proteins myoglobin (HMb) and horseradish peroxidase (HRP) with zinc oxide (ZnO) nanoparticles are investigated by using UV-vis absorption, steady state fluorescence, synchronous fluorescence, time-resolved fluorescence, FT-IR, atomic force microscopy (AFM) and circular dichroism (CD) techniques under physiological condition of pH˜7.4. The presence of mainly static mode in fluorescence quenching mechanism of HMb and HRP by ZnO nanoparticle indicates the possibility of formation of ground state complex. The processes of bindings of ZnO nanoparticles with the two proteins are spontaneous molecular interaction procedures. In both cases hydrogen bonding plays a major role. The circular dichroism (CD) spectra reveal that a helicity of the proteins is reduced by increasing ZnO nanoparticle concentration although the α-helical structures of HMb and HRP retain their identity. On binding to the ZnO nanoparticles the secondary structure of HRP molecules (or HMb molecules) remains unchanged while there is a substantial change in the environment of the tyrosin active site in case of HRP molecules and tryptophan active site in case of HMb molecules. Tapping mode atomic force microscopy (AFM) was applied for the investigation the structure of HRP adsorbed in the environment of nanoparticles on the silicon and on the bare silicon. HRP molecules adsorb and aggregate on the mica with ZnO nanoparticle. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed on the bare silicon wafer. The adsorption of HRP in the environment of ZnO nanoparticle changes drastically the domains due to a strong interaction between HRP and ZnO nanoparticles. Similar situation is observed in case of HMb molecules. These findings demonstrate the efficacy of biomedical applications of ZnO nanoparticles as well as in elucidating their mechanisms of action as drugs in both human and plant systems.

Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

PubMed

Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M; Hiasa, Hiroshi; Marks, Kevin R; Kerns, Robert J; Berger, James M; Drlica, Karl

2014-05-02

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

Molecular docking studies on tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepinone (TIBO) derivatives as HIV-1 NNRT inhibitors

NASA Astrophysics Data System (ADS)

Sapre, Nitin S.; Gupta, Swagata; Pancholi, Nilanjana; Sapre, Neelima

2008-02-01

At present, chemotherapy seems to be the main weapon in the arsenal of remedies for the ongoing crusade against AIDS. The mode of binding of the TIBO family of inhibitors has been of interest because these compounds do not fit the two-hinged-ring model as generally observed in the NNRTIs. Flexible docking simulations were performed with a series of 53 TIBO derivatives as NNRTIs. Binding preferences as well as the structural and energetic factors associated with them were studied. A good correlation ( r 2 = 0.849, q 2 = 0.843) was observed between the biological activity and binding affinity of the compounds which suggest that the identified binding conformations of these inhibitors are reliable. Further screening of PubChem database yielded novel scaffolds. Our studies suggest that modifications to the TIBO group of inhibitors might enhance their binding efficacy and hence, potentially, their therapeutic utility.

How Proteins Bind Macrocycles

PubMed Central

Villar, Elizabeth A.; Beglov, Dmitri; Chennamadhavuni, Spandan; Porco, John A.; Kozakov, Dima; Vajda, Sandor; Whitty, Adrian

2014-01-01

The potential utility of synthetic macrocycles as drugs, particularly against low druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles can expand the number of proteins that are druggable by synthetic small molecules. PMID:25038790

Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

PubMed Central

Fang, Pengfei; Guo, Min

2015-01-01

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories. PMID:26670257

Molecular design of two sterol 14α-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole

NASA Astrophysics Data System (ADS)

Rupp, Bernd; Raub, Stephan; Marian, Christel; Höltje, Hans-Dieter

2005-03-01

Sterol 14α-demethylase (CYP51) is one of the known major targets for azole antifungals. Therapeutic side effects of these antifungals are based on interactions of the azoles with the human analogue enzyme. This study describes for the first time a comparison of a human CYP51 (HU-CYP51) homology model with a homology model of the fungal CYP51 of Candida albicans (CA-CYP51). Both models are constructed by using the crystal structure of Mycobacterium tuberculosis MT-CYP51 (PDB code: 1EA1). The binding mode of the azole ketoconazole is investigated in molecular dynamics simulations with the GROMACS force field. The usage of special parameters for the iron azole complex binding is necessary to obtain the correct complex geometry in the active site of the enzyme models. Based on the dynamics simulations it is possible to explain the enantioselectivity of the human enzyme and also to predict the binding mode of the isomers of ketoconazole in the active site of the fungal model.

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

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

Wong, Jaslyn E. M. M.; Midtgaard, Søren Roi; Gysel, Kira

The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of themore » Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.« less

Predicted Structures of the Proton-Bound Membrane-Embedded Rotor Rings of the Saccharomyces cerevisiae and Escherichia coli ATP Synthases.

PubMed

Zhou, Wenchang; Leone, Vanessa; Krah, Alexander; Faraldo-Gómez, José D

2017-04-20

Recent years have witnessed a renewed interest in the ATP synthase as a drug target against human pathogens. Indeed, clinical, biochemical, and structural data indicate that hydrophobic inhibitors targeting the membrane-embedded proton-binding sites of the c-subunit ring could serve as last-resort antibiotics against multidrug resistant strains. However, because inhibition of the mitochondrial ATP synthase in humans is lethal, it is essential that these inhibitors be not only potent but also highly selective for the bacterial enzyme. To this end, a detailed understanding of the structure of this protein target is arguably instrumental. Here, we use computational methods to predict the atomic structures of the proton-binding sites in two prototypical c-rings: that of the ATP synthase from Saccharomyces cerevisiae, which is a model system for mitochondrial enzymes, and that from Escherichia coli, which can be pathogenic for humans. Our study reveals the structure of these binding sites loaded with protons and in the context of the membrane, that is, in the state that would mediate the recognition of a potential inhibitor. Both structures reflect a mode of proton coordination unlike those previously observed in other c-ring structures, whether experimental or modeled.

Structural and functional features of lysine acetylation of plant and animal tubulins.

PubMed

Rayevsky, Alexey V; Sharifi, Mohsen; Samofalova, Dariya A; Karpov, Pavel A; Blume, Yaroslav B

2017-10-10

The study of the genome and the proteome of different species and representatives of distinct kingdoms, especially detection of proteome via wide-scaled analyses has various challenges and pitfalls. Attempts to combine all available information together and isolate some common features for determination of the pathway and their mechanism of action generally have a highly complicated nature. However, microtubule (MT) monomers are highly conserved protein structures, and microtubules are structurally conserved from Homo sapiens to Arabidopsis thaliana. The interaction of MT elements with microtubule-associated proteins and post-translational modifiers is fully dependent on protein interfaces, and almost all MT modifications are well described except acetylation. Crystallography and interactome data using different approaches were combined to identify conserved proteins important in acetylation of microtubules. Application of computational methods and comparative analysis of binding modes generated a robust predictive model of acetylation of the ϵ-amino group of Lys40 in α-tubulins. In turn, the model discarded some probable mechanisms of interaction between elements of interest. Reconstruction of unresolved protein structures was carried out with modeling by homology to the existing crystal structure (PDBID: 1Z2B) from B. taurus using Swiss-model server, followed by a molecular dynamics simulation. Docking of the human tubulin fragment with Lys40 into the active site of α-tubulin acetyltransferase, reproduces the binding mode of peptidomimetic from X-ray structure (PDBID: 4PK3). © 2017 International Federation for Cell Biology.

Metal-Ion Effects on the Polarization of Metal-Bound Water and Infrared Vibrational Modes of the Coordinated Metal Center of Mycobacterium tuberculosis Pyrazinamidase via Quantum Mechanical Calculations

PubMed Central

2014-01-01

Mycobacterium tuberculosis pyrazinamidase (PZAse) is a key enzyme to activate the pro-drug pyrazinamide (PZA). PZAse is a metalloenzyme that coordinates in vitro different divalent metal cofactors in the metal coordination site (MCS). Several metals including Co2+, Mn2+, and Zn2+ are able to reactivate the metal-depleted PZAse in vitro. We use quantum mechanical calculations to investigate the Zn2+, Fe2+, and Mn2+ metal cofactor effects on the local MCS structure, metal–ligand or metal–residue binding energy, and charge distribution. Results suggest that the major metal-dependent changes occur in the metal–ligand binding energy and charge distribution. Zn2+ shows the highest binding energy to the ligands (residues). In addition, Zn2+ and Mn2+ within the PZAse MCS highly polarize the O–H bond of coordinated water molecules in comparison with Fe2+. This suggests that the coordination of Zn2+ or Mn2+ to the PZAse protein facilitates the deprotonation of coordinated water to generate a nucleophile for catalysis as in carboxypeptidase A. Because metal ion binding is relevant to enzymatic reaction, identification of the metal binding event is important. The infrared vibrational mode shift of the C=Nε (His) bond from the M. tuberculosis MCS is the best IR probe to metal complexation. PMID:25055049

Guiding, bending, and splitting of coupled defect surface modes in a surface-wave photonic crystal

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

Gao, Zhen; Gao, Fei; Zhang, Baile, E-mail: blzhang@ntu.edu.sg

2016-01-25

We experimentally demonstrate a type of waveguiding mechanism for coupled surface-wave defect modes in a surface-wave photonic crystal. Unlike conventional spoof surface plasmon waveguides, waveguiding of coupled surface-wave defect modes is achieved through weak coupling between tightly localized defect cavities in an otherwise gapped surface-wave photonic crystal, as a classical wave analogue of tight-binding electronic wavefunctions in solid state lattices. Wave patterns associated with the high transmission of coupled defect surface modes are directly mapped with a near-field microwave scanning probe for various structures including a straight waveguide, a sharp corner, and a T-shaped splitter. These results may find usemore » in the design of integrated surface-wave devices with suppressed crosstalk.« less

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

PubMed Central

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

2013-01-01

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

Exploring the selectivity of auto-inducer complex with LuxR using molecular docking, mutational studies and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Rajamanikandan, Sundaraj; Srinivasan, Pappu

2017-03-01

Bacteria communicate with one another using extracellular signaling molecules called auto-inducers (AHLs), a process termed as quorum sensing. The quorum sensing process allows bacteria to regulate various physiological activities. In this regard, quorum sensing master regulator LuxR from Vibrio harveyi represents an attractive therapeutic target for the development of novel anti-quorum sensing agents. Eventhough the binding of AHL complex with LuxR is evidenced in earlier reports, but their mode of binding is not clearly determined. Therefore, in the present work, molecular docking, in silico mutational studies, molecular dynamics simulations and free energy calculations were performed to understand the selectivity of AHL into the binding site of LuxR. The results revealed that Asn133 and Gln137 residues play a crucial role in recognizing AHL more effectively into the binding site of LuxR with good binding free energy. In addition to that, the carbonyl group presents in the lactone ring and amide group of AHL plays a vital role in the formation of hydrogen bond interactions with the protein. Further, structure based virtual screening was performed using ChemBridge database to screen potent lead molecules against LuxR. 4-benzyl-2-pyrrolidinone and N-[2(1-cyclohexen-1-yl) enthyl]-N'(2-ethoxyphenyl) were selected based on dock score, binding affinity and mode of interactions with the receptor. Furthermore, binding free energy, density functional theory and ADME prediction were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-quorum sensing drugs.

The ATP-binding site of type II topoisomerases as a target for antibacterial drugs.

PubMed

Maxwell, Anthony; Lawson, David M

2003-01-01

DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.

Structural basis of IFNα receptor recognition by TYK2

PubMed Central

Wallweber, Heidi J.A.; Tam, Christine; Franke, Yvonne; Starovasnik, Melissa A.; Lupardus, Patrick J.

2014-01-01

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family of non-receptor tyrosine kinases, which are essential for proper signaling in immune responses and development. Here we present a 2.0 angstrom resolution crystal structure of a receptor-binding fragment of human TYK2 encompassing the FERM and SH2 domains in complex with a so-called “box2” containing intracellular peptide motif from the IFNα receptor (IFNAR1). The TYK2–IFNAR1 interface reveals an unexpected receptor-binding mode that mimics a SH2 domain–phosphopeptide interaction, with a glutamate replacing the canonical phosphotyrosine residue. This structure provides the first view to our knowledge of a JAK in complex with its cognate receptor and defines the molecular logic through which JAKs evolved to interact with divergent receptor sequences. PMID:24704786

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

PubMed Central

Cai, M.; Huang, Y.; Caffrey, M.; Zheng, R.; Craigie, R.; Clore, G. M.; Gronenborn, A. M.

1998-01-01

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex. PMID:9865962

Virtual interface substructure synthesis method for normal mode analysis of super-large molecular complexes at atomic resolution.

PubMed

Chen, Xuehui; Sun, Yunxiang; An, Xiongbo; Ming, Dengming

2011-10-14

Normal mode analysis of large biomolecular complexes at atomic resolution remains challenging in computational structure biology due to the requirement of large amount of memory space and central processing unit time. In this paper, we present a method called virtual interface substructure synthesis method or VISSM to calculate approximate normal modes of large biomolecular complexes at atomic resolution. VISSM introduces the subunit interfaces as independent substructures that join contacting molecules so as to keep the integrity of the system. Compared with other approximate methods, VISSM delivers atomic modes with no need of a coarse-graining-then-projection procedure. The method was examined for 54 protein-complexes with the conventional all-atom normal mode analysis using CHARMM simulation program and the overlap of the first 100 low-frequency modes is greater than 0.7 for 49 complexes, indicating its accuracy and reliability. We then applied VISSM to the satellite panicum mosaic virus (SPMV, 78,300 atoms) and to F-actin filament structures of up to 39-mer, 228,813 atoms and found that VISSM calculations capture functionally important conformational changes accessible to these structures at atomic resolution. Our results support the idea that the dynamics of a large biomolecular complex might be understood based on the motions of its component subunits and the way in which subunits bind one another. © 2011 American Institute of Physics

Unexpected Binding Mode of a Potent Indeno[1,2-b]indole-Type Inhibitor of Protein Kinase CK2 Revealed by Complex Structures with the Catalytic Subunit CK2α and Its Paralog CK2α′

PubMed Central

Hochscherf, Jennifer; Lindenblatt, Dirk; Witulski, Benedict; Birus, Robin; Aichele, Dagmar

2017-01-01

Protein kinase CK2, a member of the eukaryotic protein kinase superfamily, is associated with cancer and other human pathologies and thus an attractive drug target. The indeno[1,2-b]indole scaffold is a novel lead structure to develop ATP-competitive CK2 inhibitors. Some indeno[1,2-b]indole-based CK2 inhibitors additionally obstruct ABCG2, an ABC half transporter overexpressed in breast cancer and co-responsible for drug efflux and resistance. Comprehensive derivatization studies revealed substitutions of the indeno[1,2-b]indole framework that boost either the CK2 or the ABCG2 selectivity or even support the dual inhibition potential. The best indeno[1,2-b]indole-based CK2 inhibitor described yet (IC50 = 25 nM) is 5-isopropyl-4-(3-methylbut-2-enyl-oxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p). Herein, we demonstrate the membrane permeability of 4p and describe co-crystal structures of 4p with CK2α and CK2α′, the paralogs of human CK2 catalytic subunit. As expected, 4p occupies the narrow, hydrophobic ATP site of CK2α/CK2α′, but surprisingly with a unique orientation: its hydrophobic substituents point towards the solvent while its two oxo groups are hydrogen-bonded to a hidden water molecule. An equivalent water molecule was found in many CK2α structures, but never as a critical mediator of ligand binding. This unexpected binding mode is independent of the interdomain hinge/helix αD region conformation and of the salt content in the crystallization medium. PMID:29236079

Structural Analysis of DFG-in and DFG-out Dual Src-Abl Inhibitors Sharing a Common Vinyl Purine Template

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

Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng

2010-09-30

Bcr-Abl is the oncogenic protein tyrosine kinase responsible for chronic myeloid leukemia (CML). Treatment of the disease with imatinib (Gleevec) often results in drug resistance via kinase mutations at the advanced phases of the disease, which has necessitated the development of new mutation-resistant inhibitors, notably against the T315I gatekeeper mutation. As part of our efforts to discover such mutation resistant Abl inhibitors, we have focused on optimizing purine template kinase inhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are also potent Src inhibitors. Here we present crystal structures of Abl bound by twomore » such inhibitors, based on a common N9-arenyl purine, and that represent both DFG-in and -out binding modes. In each structure the purine template is bound deeply in the adenine pocket and the novel vinyl linker forms a non-classical hydrogen bond to the gatekeeper residue, Thr315. Specific template substitutions promote either a DFG-in or -out binding mode, with the kinase binding site adjusting to optimize molecular recognition. Bcr-Abl T315I mutant kinase is resistant to all currently marketed Abl inhibitors, and is the focus of intense drug discovery efforts. Notably, our DFG-out inhibitor, AP24163, exhibits modest activity against this mutant, illustrating that this kinase mutant can be inhibited by DFG-out class inhibitors. Furthermore our DFG-out inhibitor exhibits dual Src-Abl activity, absent from the prototypical DFG-out inhibitor, imatinib as well as its analog, nilotinib. The data presented here provides structural guidance for the further design of novel potent DFG-out class inhibitors against Src, Abl and Abl T315I mutant kinases.« less