Homology modeling of human CCR5 and analysis of its binding properties through molecular docking and molecular dynamics simulation

In this study, homology modeling, molecular docking and molecular dynamics simulation were performed to explore structural features and binding mechanism of some inhibitors of chemokine receptor type 5 (CCR5), and to construct a model for designing new CCR5 inhibitors for preventing HIV attachment to the host cell. A homology modeling procedure was employed to construct a 3D model of CCR5. For this procedure, the X-ray crystal structure of bovine rhodopsin (1F88A) at 2.80? resolution was used as template. After inserting the constructed model into a hydrated lipid bilayer, a 20ns molecular dynamics (MD) simulation was performed on the whole system. After reaching the equilibrium, twenty-four CCR5 inhibitors were docked in the active site of the obtained model. The binding models of the investigated antagonists indicate the mechanism of binding of the studied compounds to the CCR5 obviously. Moreover, 3D pictures of inhibitor-protein complex provided precious data regarding the binding orientation of each antagonist into the active site of this protein. One additional 20 ns MD simulation was performed on the initial structure of the CCR5-ligand 21 complex, resulted from the previous docking calculations, embedded in a hydrated POPE bilayer to explore the effects of the presence of lipid bilayer in the vicinity of CCR5-ligand complex. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.     

Unbiased simulations reveal the inward-facing conformation of the human serotonin transporter and Na(+) ion release

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 μs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na(+)-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport.     

Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn2+-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn2+-binding sites between V102 or W103 (TMH I) and I179-L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn2+ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn2+-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.     

Molecular dynamics simulation of a human thiopurine S-methyltransferase complexed with 6-mercaptopurine model

Human thiopurine S-methyltransferase (TPMT) is an essential protein in 6-mercaptopurine (6MP) drug metabolism. To understand the pharmacogenetics of TPMT and 6MP, X-ray co-crystal structures of TPMT complexes with S-adenosyl-L-methionine (AdoMet) and 6MP are required. However, the co-crystal structure of this complex has not been reported because 6MP is poorly water soluble. We used molecular dynamics (MD) simulation to predict the structure of the complex of human TPMT-AdoHcy(CH₂)6MP, where the sulfur atoms of AdoHcy and 6MP were linked by a CH₂ group. After 1300 picoseconds of MD simulation, the trajectory showed that 6MP was stabilized in the TPMT active site by formation of non-bonded interactions between 6MP and Phe40, Pro196 and Arg226 side chains of TPMT. The intersulfur distance between AdoHcy and 6MP as well as the binding modes and the interactions of our TPMT-AdoHcy model are consistent with those observed in the X-ray crystal structure of murine TPMT-AdoHcy-6MP complex. The predicted binding modes of AdoHcy and 6MP in our model are consistent with those observed in murine TPMT X-ray crystal structures, which provides structural insights into the interactions of TPMT, AdoHcy, and 6MP at the atomic level and may be used as a starting point for further study of thiopurine drug pharmacogenetics.     

Computational modeling of human coreceptor CCR5 antagonist as a HIV-1 entry inhibitor: using an integrated homology modeling,docking, and membrane molecular dynamics simulation analysis approach

Chemokine receptor 5 (CCR5) is an integral membrane protein that is utilized during human immunodeficiency virus type-1 entry into host cells. CCR5 is a G-protein coupled receptor that contains seven transmembrane (TM) helices. However, the crystal structure of CCR5 has not been reported. A homology model of CCR5 was developed based on the recently reported CXCR4 structure as template. Automated docking of the most potent (14), medium potent (37), and least potent (25) CCR5 antagonists was performed using the CCR5 model. To characterize the mechanism responsible for the interactions between ligands (14, 25, and 37) and CCR5, membrane molecular dynamic (MD) simulations were performed. The position and orientation of ligands (14, 25, and 37) were found to be changed after MD simulations, which demonstrated the ability of this technique to identify binding modes. Furthermore, at the end of simulation, it was found that residues identified by docking were changed and some new residues were introduced in the proximity of ligands. Our results are in line with the majority of previous mutational reports. These results show that hydrophobicity is the determining factor of CCR5 antagonism. In addition, salt bridging and hydrogen bond contacts between ligands (14, 25, and 37) and CCR5 are also crucial for inhibitory activity. The residues newly identified by MD simulation are Ser160, Phe166, Ser180, His181, and Trp190, and so far no site-directed mutagenesis studies have been reported. To determine the contributions made by these residues, additional mutational studies are suggested. We propose a general binding mode for these derivatives based on the MD simulation results of higher (14), medium (37), and lower (25) potent inhibitors. Interestingly, we found some trend for these inhibitors such as, salt bridge interaction between basic nitrogen of ligand and acidic Glu283 seemed necessary for inhibitory activity. Also, two aromatic pockets (pocket I – TM1-3 and pocket II – TM3-6) were linked by the central polar region in TM7, and the simulated inhibitors show important interactions with the Trp86, Tyr89, Tyr108, Phe112, Ile198, Tyr251, Leu255, and Gln280 and Glu283 residues. These results shed light on the usage of MD simulation to identify more stable, optimal binding modes of the inhibitors.     

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

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 (LeuT(Aa)) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) 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 critically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) 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 A of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.     

Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 Å above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp⁹⁸. Furthermore, the 7-position of the imipramine ring is found vicinal to Phe³³⁵, and the pocket lined by Ala¹⁷³ and Thr⁴³⁹ is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).     

Homology modeling,molecular dynamic simulation and docking studies of cyclin dependent kinase 1

In order to develop promising cyclin dependent kinase 1 inhibitors, homology modeling, docking and molecular dynamic simulation techniques were applied to get insight into the functional and structural properties of cyclin dependent kinase 1 (CDK1). Since there is no reported CDK1 crystal structural data, the three dimensional structure of CDK1 was constructed based on homology modeling. An extensive dynamic simulation was also performed on a Flavopiridol-CDK1 complex for probing the binding pattern of Flavopiridol in the active site of CDK1. The binding modes of other inhibitors to CDK1 were also proposed by molecular docking. The structural requirement for developing more potent CDK1 inhibitors was obtained by the above-mentioned molecular simulations and pharmacophore modeling.     

Structure and dynamics of the full-length M1 muscarinic acetylcholine receptor studied by molecular dynamics simulations

The three-dimensional structure of full-length structure of the M₁ muscarinic receptor was obtained through the fragmental homology modeling procedure. A 10-ns molecular dynamics (MD) simulation of the protein imbedded in a lipid slab and surrounded by water molecules was further used to relax the model. It was found that the homology model corresponded to the conformation in the ground state, since no significant motions of the backbone of transmembrane domains were observed. Furthermore, the reliability of the model was validated by analyzing key inter-helical contacts, sidechain-sidechain interactions, the formation of stable aromatic microdomains (clusters) and the docking of acetylcholine to its binding site. Moreover, a few conserved interactions observed in the X-ray structure of rhodopsin, such as inter-helical sidechain-sidechain hydrogen bonds were accurately reproduced in the MD simulation. The coupling of ACh to its binding site was found to be dominated by π-cation and salt bridge interactions, while its conformational space was restrained through van der Waals and hydrogen bond interactions. In general, such features were in very good agreement with the available experimental as well as with theoretical data. Considering the above, the structural information obtained in this study can be used a starting point to investigate the activation mechanism of the receptor and the ability to develop selective agonists and allosteric modulators which could be used for the treatment of Alzheimer’s disease.     

Molecular dynamics simulation and molecular docking studies of 1,4-Dihydropyridines as P-glycoprotein’s allosteric inhibitors

P-glycoprotein (P-gp) is a main factor contributing to multidrug resistance. The effect of this transporter protein on limiting the effectiveness of chemotherapy has been shown by various studies. In a previous report, we synthesized some 14-dihydropyridine (DHP) derivatives as inhibitors of human P-gp. In the present study, a computational approach has been exploited to reveal the main interactions between DHPs and P-gp. In order to do this, homology modeling was performed to obtain a model of the protein. Then, molecular dynamics simulation was used to refine the constructed model of P-gp in the presence of the lipids bilayer. Model validation was performed with several tools. Finally, molecular docking followed by MD simulation of ligand–protein complex was employed to elucidate the binding mode and the dynamical changes of protein with/without DHPs bound. The results emphasized that interaction of the residues Gln912, Ser909, Arg905, Ser474, Val472 with DHPs play a crucial role in the inhibitory of these ligands and this was in a relatively good accordance with the results reported in the experimental studies.     

Homology modeling and examination of the effect of the D92E mutation on the H5N1 nonstructural protein NS1 effector domain

Virulent H5N1 strains of influenza virus often harbor a D92E point mutation in the nonstructural protein NS1. This crucial mutation has been correlated with increased virulence and/or cytokine resistance, but the structural implications of such a change are still unclear. Furthermore, NS1 protein could also be a potential target for the development of novel antiviral agents against H5N1 strains. Therefore, a reasonable 3D model of H5N1 NS1 is important for the understanding of the molecular basis of increased virulence and the design of novel antiviral agents. Based on the crystal structure of a non-H5N1 NS1 protein, a model of H5N1 NS1 was developed by homology modeling, molecular mechanics and molecular dynamics simulations. It was found that the D92E mutation could result in weakened interactions of the carboxylate side chain with other phosphorylated residues, thereby activating phosphorylation of NS1. Figure Superposition of snapshots picked from the two molecular dynamic (MD) trajectories: a H5N1 NS1 homology model and b non-H5N1 NS1 crystal structure after 0 (green ribbon), 5 (blue ribbon) and 10 ns (pink ribbon) MD simulation     

Atomistic models of ion and solute transport by the sodium-dependent secondary active transporters

The recent determination of high-resolution crystal structures of several transporters offers unprecedented insights into the structural mechanisms behind secondary transport. These proteins utilize the facilitated diffusion of the ions down their electrochemical gradients to transport the substrate against its concentration gradient. The structural studies revealed striking similarities in the structural organization of ion and solute binding sites and a well-conserved inverted-repeat topology between proteins from several gene families. In this paper we will overview recent atomistic simulations applied to study the mechanisms of selective binding of ion and substrate in LeuT, Glt, vSGLT and hSERT as well as its consequences for the transporter conformational dynamics. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Structure and atomic fluctuation patterns of potato carboxypeptidase a inhibitor protein

Molecular dynamics (MD) simulation methods were applied to the study of the structural and dynamic fluctuation properties of the potato carboxypeptidase A inhibitor protein (PCI) immersed in a bath of 1259 water molecules. A trajectory of 200 ps was generated at constant temperature and pressure. The crystallographic structure of PCI, as found in its complex with bovine carboxy-peptidase A (CPA), was used to seed the MD simulation. Analyses show that the structure of the PCI core is fairly rigid and stable in itself, and that little deformation is caused by the protein-protein interactions found in the PCI-CPA complex. The N-terminal tail fluctuates to approach the core structure and appears as a relatively disordered region. In contrast, the conformations of the C-terminal tail, which is involved in the inhibitory mechanism, fluctuates in the neighborhood of the X-ray structure in orientations which facilitate CPA binding. Comparison with the structural entries for PCI in water obtained from both 2D-NMR experiments and X-ray data shows that important features of the MD structural results fluctuates between the initial crystal values and those obtained from the NMR solution structure. This fluctuation is not uniform; minor regions move away from the X-ray conformation while they do not approach the NMR conformation. The results reported suggest that the trajectory is long enough to show a behavior that is consistent with the conformational space available to the protein in solution.Abbreviations CPA Carboxypeptidase - DG Distance Geometry - NMR Nuclear Magnetic Resonance - NIS Non Inertial Solvent - MD Molecular Dynamics - PBC Periodic Boundary Conditions - PCI Potato Carboxypeptidase Inhibitor - RMSD Root Mean Square Deviation - a.m.u. Atomic mass units Correspondence to: O. Tapia     

Investigation of ligand selectivity in CYP3A7 by molecular dynamics simulations

Cytochrome P450 (CYP) 3A7 plays a crucial role in the biotransformation of the metabolized endogenous and exogenous steroids. To compare the metabolic capabilities of CYP3A7–ligands complexes, three endogenous ligands were selected, namely dehydroepiandrosterone (DHEA), estrone, and estradiol. In this study, a three-dimensional model of CYP3A7 was constructed by homology modeling using the crystal structure of CYP3A4 as the template and refined by molecular dynamics simulation (MD). The docking method was adopted, combined with MD simulation and the molecular mechanics generalized born surface area method, to probe the ligand selectivity of CYP3A7. These results demonstrate that DHEA has the highest binding affinity, and the results of the binding free energy were in accordance with the experimental conclusion that estrone is better than estradiol. Moreover, several key residues responsible for substrate specificity were identified on the enzyme. Arg372 may be the most important residue due to the low interaction energies and the existence of hydrogen bond with DHEA throughout simulation. In addition, a cluster of Phe residues provides a hydrophobic environment to stabilize ligands. This study provides insights into the structural features of CYP3A7, which could contribute to further understanding of related protein structures and dynamics.     

Molecular dynamics simulations and membrane protein structure quality

Despite a growing repertoire of membrane protein structures (currently ∼120 unique structures), considerations of low resolution and crystallization in the absence of a lipid bilayer require the development of techniques to assess the global quality of membrane protein folds. This is also the case for assessment of, e.g. homology models of human membrane proteins based on structures of (distant) bacterial homologues. Molecular dynamics (MD) simulations may be used to help evaluate the quality of a membrane protein structure or model. We have used a structure of the bacterial ABC transporter MsbA which has the correct transmembrane helices but an incorrect handedness and topology of their packing to test simulation methods of quality assessment. An MD simulation of the MsbA model in a lipid bilayer is compared to a simulation of another bacterial ABC transporter, BtuCD. The latter structure has demonstrated good conformational stability in the same bilayer environment and over the same timescale (20 ns) as for the MsbA model simulation. A number of comparative analyses of the two simulations were performed to assess changes in the structural integrity of each protein. The results show a significant difference between the two simulations, chiefly due to the dramatic structural deformations of MsbA. We therefore propose that MD could become a useful quality control tool for membrane protein structural biology. In particular, it provides a way in which to explore the global conformational stability of a model membrane protein fold.     

Structure prediction and molecular dynamics simulations of a G-protein coupled receptor: human CCR2 receptor

CC chemokine receptor type-2 (CCR2) is a member of G-protein coupled receptors superfamily, expressed on the cell surface of monocytes and macrophages. It binds to the monocyte chemoattractant protein-1, a CC chemokine, produced at the sites of inflammation and infection. A homology model of human CCR2 receptor based on the recently available C-X-C chemokine recepor-4 crystal structure has been reported. Ligand information was used as an essential element in the homology modeling process. Six known CCR2 antagonists were docked into the model using simple and induced fit docking procedure. Docked complexes were then subjected to visual inspection to check their suitability to explain the experimental data obtained from site directed mutagenesis and structure-activity relationship studies. The homology model was refined, validated, and assessed for its performance in docking-based virtual screening on a set of CCR2 antagonists and decoys. The docked complexes of CCR2 with the known antagonists, TAK779, a dual CCR2/CCR5 antagonist, and Teijin-comp1, a CCR2 specific antagonist were subjected to molecular dynamics (MD) simulations, which further validated the binding modes of these antagonists. B-factor analysis of 20?ns MD simulations demonstrated that Cys190 is helpful in providing structural rigidity to the extracellular loop (EL2). Residues important for CCR2 antagonism were recognized using free energy decomposition studies. The acidic residue Glu291 from TM7, a conserved residue in chemokine receptors, is favorable for the binding of Teijin-comp1 with CCR2 by ΔG of ?11.4?kcal/mol. Its contribution arises more from the side chains than the backbone atoms. In addition, Tyr193 from EL2 contributes ?0.9?kcal/mol towards the binding of the CCR2 specific antagonist with the receptor. Here, the homology modeling and subsequent molecular modeling studies proved successful in probing the structure of human CCR2 chemokine receptor for the structure-based virtual screening and predicting the binding modes of CCR2 antagonists.     

Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations

BackgroundTim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution.MethodsThe conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations.ResultsMD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop.ConclusionsOur work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins.General significanceComputational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins.     

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.     

Molecular modeling aided design of nicotinic acid receptor GPR109A agonists

A homology model of the nicotinic acid receptor GPR109A was constructed based on the X-ray crystal structure of bovine rhodopsin. An HTS hit was docked into the homology model. Characterization of the binding pocket by a grid-based surface calculation of the docking model suggested that a larger hydrophobic body plus a polar tail would improve interaction between the ligand and the receptor. The designed compounds were synthesized, and showed significantly improved binding affinity and activation of GPR109A.