Structure Of Bovine Rhodopsin Research Articles

Graph-based algorithms to dissect long-distance water-mediated H-bond networks for conformational couplings in GPCRs.

Changes in structure and dynamics elicited by agonist ligand binding at the extracellular side of G protein coupled receptors (GPCRs) must be relayed to the cytoplasmic G protein binding side of the receptors. To decipher the role of water-mediated hydrogen-bond networks in this relay mechanism, we have developed graph-based algorithms and analysis methodologies applicable to datasets of static structures of distinct GPCRs. For a reference dataset of static structures of bovine rhodopsin solved at the same resolution, we show that graph analyses capture the internal protein-water hydrogen-bond network. The extended analyses of static structures of rhodopsins and opioid receptors suggest a relay mechanism whereby inactive receptors have in place much of the internal core hydrogen-bond network required for long-distance relay of structural change, with extensive local H-bond clusters observed in structures solved at high resolution and with internal water molecules.

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery.

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.

Coumarinyl pyranopyrimidines as new neuropeptide S receptor antagonists; design, synthesis, homology and molecular docking

In this work, we described the design, synthesis and characterization of a new class of NPSR antagonists bearing the tetracyclic coumarinyl pyranopyrimidine scaffold incorporated with different acyclic and/or heterocyclic moieties. These compounds are highlighted in this study as never being used as NPSR antagonists before which provides a model for the discovery of new bioactive inhibitors that may hold potential for drug development towards anxiety, food, and addiction disorders. Synthetic and medicinal chemistry studies led to the identification of four potent antagonists, compounds 7d, 10, 12 and 13, which were able to significantly inhibit the stimulatory effect of NPS through counteracting the increased intracellular Ca2+ accumulation. The target compound 7d was the most active derivative behaving as a pure NPSR antagonist and displaying IC50 value of 2 μM. Homology model of NPSR was built based on bovine rhodopsin structure. Modeling studies were carried out to further rationalize the NPSR binding mode of the target compounds. Moreover, molecular dynamics simulation study was performed for compounds 7d, 10 and 12 which revealed the stability of the ligand-protein complex and the reliability of the docking studies.

In Silico Investigations of Possible Therapeutic Peptides as Drugs for Anti-Inflammatory Responses

The chemokine called interleukin-8 (IL-8 or CXCL8) plays a central role in human immune response by binding and activating the receptor CXCR1 that belongs to the G-protein coupled receptor (GPCR) family. Upon binding of CXCL8, CXCR1 undergoes conformational change resulting in signal transduction.The purpose of this study was to determine the role of the fragments of the CXCL8 binding sites of CXCR1 as potential therapeutic targets for developing novel drugs against inflammatory diseases.Homology modeling studies were firstly performed to construct the three-dimensional structure of CXCR1 by taking the crystal structure of bovine rhodopsin (PDB code: 1U19) as the template with the sequence similarity up to 41.8 %. By the following molecular docking programs several potential peptides were selected and synthesized to validate the anti-inflammatory efficiency through flow cytometry analysis. The preferred complexes of CXCL8 binding CXCR1 from docking predictions were then embedded into POPC lipid bilayers for 50 ns MD simulation to investigate the binding interaction. Simulation results show that electrostatic interaction dominates the binding of CXCL8 and CXCR1, consistent with the previous experiments.Small peptide drugs are novel therapeutic candidates for inflammatory diseases. High-throughput, structure-based virtual screening combining MD simulations is an effective computer-based drug design method for discovering anti-inflammatory drug candidates.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Modeling and Simulation Studies of Human β3 Adrenergic Receptor and its Interactions with Agonists

β3 adrenergic receptor (β3AR) is known to mediate various pharmacological and physiological effects such as thermogenesis in brown adipocytes, lipolysis in white adipocytes, glucose homeostasis and intestinal smooth muscle relaxation. Several efforts have been made in this field to understand their function and regulation in different human tissues and they have emerged as potential attractive targets in drug discovery for the treatment of diabetes, depression, obesity etc. Although the crystal structures of Bovine Rhodopsin and β2 adrenergic receptor have been resolved, to date there is no three dimensional structural information on β3AR. Our aim in this study was to model 3D structure of β3AR by various molecular modeling and simulation techniques. In this paper, we describe a refined predicted model of β3AR using different algorithms for structure prediction. The structural refinement and minimization of the generated 3D model of β3AR were done by Schrodinger suite 9.1. Docking studies of β3AR model with the known agonists enabled us to identify specific residues, viz, Asp 117, Ser 208, Ser 209, Ser 212, Arg 315, Asn 332, within the β3AR binding pocket, which might play an important role in ligand binding. Receptor ligand interaction studies clearly indicated that these five residues showed strong hydrogen bonding interactions with the ligands. The results have been correlated with the experimental data available. The predicted ligand binding interactions and the simulation studies validate the methods used to predict the 3D-structure.

Homology Modeling and Antagonist Binding Site Study of the Human Histamine H2 Receptor

On the basis of the high resolution crystal structures of bovine rhodopsin, human beta2-adrenergic receptor and human A(2a) adenosine receptor, three-dimensional structure of the human histamine H2 receptor (HHR2) was developed by homology modeling. Results of the evaluations suggest that a high quality homology model for HHR2 has been obtained. Three antagonists, cimetidine, ranitidine and nizatidine, were applied to binding site study with this model through molecular docking, molecular dynamics simulations and the molecular mechanics-Poisson-Boltzmann surface area calculations. One aspartic acid, Asp98 in transmembrane domain 7 (TM3), has been identified as major contributors to ligand binding by H-bond interactions. Asn159 in TM4 and Asp186 in TM5 are of great importance in stabilizing HHR2- antagonist complexes. Two hydrophobic sites especially two residues, Val99 in TM3 and Phe254 in TM6, were identified to be essential for their strong hydrophobic interactions with antagonists. The findings reported here are in agreement with available experimental mutagenesis data. This study should be very helpful for a better understanding of the action mode of the antagonist and for guiding further drug design and mutagenesis studies.

Building a Model of the Blue Cone Pigment Based on the Wild Type Rhodopsin Structure with QM/MM Methods

Understanding the mechanism of color tuning of the retinal chromophore by its host protein became one of the key issues in the research on rhodopsins. While early mutation studies addressed its genetic origin, recent studies advanced to investigate its structural origin, based on X-ray crystallographic structures. For the human cone pigments, no crystal structures have been produced, and homology models were employed to elucidate the origin of its blue-shifted absorption. In this theoretical study, we take a different route to establish a structural model for human blue. Starting from the well-resolved structure of bovine rhodopsin, we derive multiple mutant models by stepwise mutation and equilibration using molecular dynamics simulations in a hybrid quantum mechanics/molecular mechanics framework. Our 30fold mutant reproduces the experimental UV-vis absorption shift of 0.45 eV and provides new insights about both structural and genetic factors that affect the excitation energy. Electrostatic effects of individual amino acids and collaborative structural effects are analyzed using semiempirical (OM2/MRCI) and ab initio (SORCI) multireference approaches.

A new era for GPCR research: structures, biology and drug discovery

Cells in a living organism must communicate with each other through continuously sending and receiving messages. G-protein coupled receptors (GPCRs) are the largest family of communicating molecules at the cell surface. They transmit diverse extracellular signals, ranging from light and small chemical hormones to large peptide and protein hormones, and as such they play crucial roles in numerous physiological and pathological processes. More importantly, GPCRs are the most successful class of drug targets that are relevant to many major diseases, including cancer, heart failure, and inflammatory diseases. Over 50% of currently used drugs are targeted to GPCRs. However, these drugs target only 50–60 GPCRs, leaving the majority of human GPCRs, exceeding 800, unexplored for drug discovery. Given the prominent roles of GPCRs in biology and their successful track records as drug targets, GPCRs have become a hot frontier in basic research of life science and therapeutic discovery of translational medicines. In this special issue, there are nine exciting reviews that cover a broad scope of GPCR structures, biology, diseases, and drug discovery. Among them, four reviews are dedicated to GPCR structures. Over the last few years, we have witnessed a revolution in Class A GPCR structural biology. Rhodopsin is the founding member of the GPCR family and its signaling mechanism is a paradigm for many other GPCRs. The crystal structure of bovine rhodopsin was first solved in 2000 and since then structures of rhodopsin have been solved in several functional states, including the inactivated dark state, partially active opsin, and the fully active state that is bound with a Gαt peptide. ZHOU, MELCHER, and XU review the signaling mechanisms gained from rhodopsin structures and compare these mechanisms to other members of Class A GPCRs, most notably β2-adrenergic receptor (β2AR)1. Class B and C GPCRs distinguished themselves from Class A GPCRs by having a large extracellular domain for binding of ligands. Compared to Class A GPCRs, structure determination for the full-length receptors of other families of GPCRs is lagging behind. However, structures for a half dozen of Class B and C GPCR extracellular domains (ECDs) in complex with their respective ligands have been determined. The structures and ligand binding mechanisms of Class B and Class C GPCRs will be reviewed by the XU and LIU groups, respectively2, 3. The rapid explosion of Class A GPCR structures is owed to technology development in various stages of structure determination; including protein expression, purification, crystallization, and X-ray diffraction, for which a thorough review is provided by ZHAO and WU4. Following the reviews of GPCR structures, five reviews are focused on GPCR biology, diseases, and drug discovery. β2AR is a prototype of GPCRs, and in parallel to rhodopsin, it has been serving as a model for studying other GPCRs. WOO and XIAO review the signaling mechanisms by β-adrenergic receptor subtypes that provide a basis for developing selective biased β2 agonists for the treatment of heart failure5. Moving from heart to immune systems, YE and SUN review the role of GPCRs in inflammation processes from chemotaxisis to transcriptional regulation of inflammation programs6. Lappano and Maggiolini provide a systematic review on the relationship of cancers with various subfamilies of GPCRs, which may serve as the basis for developing novel pharmacological interventions for cancers7. A large number of GPCRs are orphan receptors whose ligand remains unknown. Despite missing cognate ligands, many orphan GPCRs are known to play important physiological functions, mostly from genetic knockout studies. LIU and colleagues provide a thorough review on different class of orphan nuclear receptors, their functions, and possible strategies for identification of endogenous ligands8. Finally, as one of the most important drug targets, GPCRs are widely pursued by both academic and industrial research for drug discovery. ZHANG and XIE provide a comprehensive review on methods of GPCR drug discovery, including various ligand binding and cell-based functional assays, many of which are tool kits for basic research as well as drug discovery for GPCRs9. Followed the waves of technological advances and inter-disciplinary approaches in modern biological research, the field of GPCRs is evolving rapidly into a new phase of exciting progresses. With a rich history of biology and drug discovery, GPCR research is expected to continue to be a dominant source of innovative medicines for the 21st Century. We hope that the collection of these nine exciting reviews will provide a window into this new era of GPCR research and drug discovery.

Prediction of Binding Mode between Chemokine Receptor CCR2 and Its Known Antagonists using Ligand Supported Homology Modeling

Chemokines play a crucial role in the trafficking of leukocytes in the body through the binding to their related receptors. Chemokine Receptor 2 (CCR2) belongs to GProtein Coupled Receptor (GPCR) family and expressed in monocytes, immature dendritic cells, activated T lymphocytes, basophils, and endothelial and vascular smooth-muscle cells. CCR2 binds several chemokines; CCL2, CCL7, CCL8, and CCL13. CCR2 and its ligand have been implicated in the pathophysiology of a number of diseases, including rheumatoid arthritis, multiple sclerosis, atherosclerosis, organ transplant rejection, and insulin resistance. Knowledge of the structural basis on CCR2-ligand interaction could help facilitate the design of novel CCR2 antagonists. We modeled and predicted the binding sites of widely known CCR2 antagonists, TAK-779 and Teijin-lead (Fig. 1), using ligand supported homology modeling method. Homology modeling predicts the three-dimensional structure of a given protein sequence based on its alignment to reference proteins of known three-dimensional structure (socalled templates). It is known as the most successful technique for predicting the three-dimensional protein structure. In conventional homology modeling method, the ligand molecules are not considered in the process of protein model building. After building the protein model, the ligand molecules are docked into the protein model. These approaches often showed the consequence that residue side chains involved in ligand binding are inappropriately modeled. The time-consuming re-modeling of the binding site or docking is required to generate sensible side-chain and ligand orientations. In the case of GPCR, the conventional homology modeling approaches for obtaining the binding site models are more challenging because the number of the available templates is very limited and the sequence identity to the templates is very low. The available templates for GPCR are the solved three-dimensional structures of bovine rhodopsin, β2-adrenergic receptor (β2AR), turkey β1adrenergic receptor, and human adenosine A2A receptor. The homology modeling from those limited number of templates may result in uncertainty not only regarding backbone positioning but also about side chain conformations. One approach to solve this is to optimize the side chains through mechanics and/or dynamics in an empty pocket; however, this method may be inaccurate due to possible disruption of the binding site. Another approach is to use dynamics and ligand data as a restraint. This method also has a possibility of poor quality. Thus, the development of modified homology modeling methods for constructing a plausible ligand binding mode is an important theme in GPCR-ligand modeling research. The incorporation of ligand information from the early stage of homology modeling has been studied as an alternative strategy to the conventional homology modeling. This approach was first developed and termed as ligand supported homology modeling by Klebe and coworkers. Recently, the improved and diversified methods have been published as ligand-steered homology modeling or homology-modeling protein-ligand interactions. The results presented reasonable binding site models. We recently reported the protocol and evaluation for the proprietary CCR2 antagonist by ligand-supported homology modeling. We here employed the protocol to predict the binding mode for known CCR2 antagonists, TAK-779 and Teijin-lead. The method in this study for structure modeling of CCR2 and its antagonists is summarized in Figure 2. In the first place of homology modeling, we retained the bound ligand, carazolol, from the crystal structure of β2AR. Then, carazolol was replaced by the various conformations of the target

Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

Molecular docking of 2-(benzimidazol-2-ylthio)-N-phenylacetamide-derived small-molecule agonists of human formyl peptide receptor 1

Human N-formyl peptide receptor 1 (FPR1) is a G protein-coupled receptor (GPCR) involved in host defense and sensing cellular damage. Since structure-based ligand design for many GPCRs, including FPR1, is restricted by the lack of experimental three dimensional structures, homology modeling has been widely used to study GPCR-ligand binding. Indeed, receptor-ligand binding mode predictions can be derived from homology modeling with supporting ligand information. In the present work, we report comparative docking studies of 2-(benzimidazol-2-ylthio)-N-phenylacetamide derived FPR1 agonists, identified here and previously, with several known FPR1 peptide agonists in a FPR1 homology model that is based on the crystal structure of bovine rhodopsin. We found that the binding pocket of the most active molecules shares some common features with high affinity FPR1 peptide agonists, suggesting that they may bind to similar binding sites. Classification tree analysis led to the derivation of a good recognition model based on four amino acid descriptors for distinguishing FPR1 ligands from inactive analogs. Hence, the corresponding residues (Thr199, Arg201, Gly202, and Ala261) can be considered as markers of important areas in the ligand binding site. Concurrently, we identified several unique binding features of benzimidazole derivatives and showed that alkoxy-substituents of the benzimidazole ring are located within a FPR1 hole bounded by Thr199, Thr265, Ile268, and Leu271 or in a groove in the vicinity of Leu198, Arg201, Gly202, and Arg205. The understanding of these molecular features will likely prove beneficial in future design of novel FPR1 agonists based on the benzimidazole scaffold.

Comparative modeling of human kappa opioid receptor and docking analysis with the peptide YFa

The kappa opioid receptor belongs to the super family of G protein – coupled receptors that are of utmost significance in the development of potent analgesic drugs for the treatment of severe pain. An accurate evaluation of the ligand binding pathways into this receptor at molecular level may play a key role in the design of new molecules with more desirable properties and reduced side effects. In this study, homology model of the human kappa opioid receptor was developed by MODELLER using the X-ray crystal structure of bovine rhodopsin as template. Initial structure of the receptor was refined computationally with energy minimization and molecular dynamics simulation at 300 K in a pre-equilibrated phospholipid bilayer by GROMACS. The Met-enkaphalin-Arg-Phe based opioid peptide YFa (YGGFMKKKFMRF) designed and characterized by our laboratory was docked into the optimized model and the critical amino acids responsible for binding were identified. A number of low energy binding poses of YFa with the receptor were assessed after the molecular docking in which the peptide was observed to interact with the receptor's extracellular amino acids through hydrogen bonds. The human kappa opioid receptor model optimized in a phospholipid bilayer should provide a good starting point for further characterization of the binding modes of other opioid ligands. Furthermore, the biologically favorable molecular interactions between YFa and human kappa opioid receptor observed by our study might be able to justify the specificity of this peptide.

Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-593⁵·⁵⁰) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular α-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences.

New Insights into the Binding Mode of Melanin Concentrating Hormone Receptor-1 Antagonists: Homology Modeling and Explicit Membrane Molecular Dynamics Simulation Study

Melanin concentrating hormone (MCH) is a cyclic 19-amino-acid peptide expressed mainly in the hypothalamus. It is involved in the control of feeding behavior, energy homeostasis, and body weight. Administration of MCH-R1 antagonists has been proved to reduce food intake and cause weight loss in animal models. In the present study, a homology model of the human MCH-R1 was constructed using the crystal structure of bovine rhodopsin (PDB: 1u19) as a template. Based on the observation that MCH-R1 can bind ligands of high chemical diversity, the initial model was subjected to an extensive ligand-supported refinement using antagonists of different chemotypes. The refinement process involved stepwise energy minimizations and molecular dynamics simulations. The refined model was inserted into a pre-equilibrated DPPC/TIP3P membrane system and then simulated for 20 ns in complex with structurally diverse antagonists. This protocol was able to explain the SAR of MCH-R1 antagonists with diverse chemical structures. Moreover, it reveals new insights into the critical recognition sites within the receptor. This work represents the first detailed study of molecular dynamics of MCH-R1 inserted into a membrane-aqueous environment.

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 2.80Å 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 20ns 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

Characterization of the β-d-Glucopyranoside Binding Site of the Human Bitter Taste Receptor hTAS2R16

G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and β-d-glucopyranoside and will also facilitate rational design of bitter blockers.

Structure-Activity Relationships of GPR120 Agonists Based on a Docking Simulation

GPR120 is a G protein-coupled receptor expressed preferentially in the intestinal tract and adipose tissue, that has been implicated in mediating free fatty acid-stimulated glucagon-like peptide-1 (GLP-1) secretion. To develop GPR120-specific agonists, a series of compounds (denoted as NCG compounds) derived from a peroxisome proliferator-activated receptor γ agonist were synthesized, and their structure-activity relationships as GPR120 agonists were explored. To examine the agonistic activities of these newly synthesized NCG compounds, and of compounds already shown to have GPR120 agonistic activity (grifolic acid and MEDICA16), we conducted docking simulation in a GPR120 homology model that was developed on the basis of a photoactivated model derived from the crystal structure of bovine rhodopsin. We calculated the hydrogen bonding energies between the compounds and the GPR120 model. These energies correlated well with the GPR120 agonistic activity of the compounds (R(2) = 0.73). NCG21, the NCG compound with the lowest calculated hydrogen bonding energy, showed the most potent extracellular signal-regulated kinase (ERK) activation in a cloned GPR120 system. Furthermore, NCG21 potently activated ERK, intracellular calcium responses and GLP-1 secretion in murine enteroendocrine STC-1 cells that express GPR120 endogenously. Moreover, administration of NCG21 into the mouse colon caused an increase in plasma GLP-1 levels. Taken together, our present study showed that a docking simulation using a GPR120 homology model might be useful to predict the agonistic activity of compounds.

FoldGPCR: structure prediction protocol for the transmembrane domain of G protein-coupled receptors from class A.

Building reliable structural models of G protein-coupled receptors (GPCRs) is a difficult task because of the paucity of suitable templates, low sequence identity, and the wide variety of ligand specificities within the superfamily. Template-based modeling is known to be the most successful method for protein structure prediction. However, refinement of homology models within 1-3 A C alpha RMSD of the native structure remains a major challenge. Here, we address this problem by developing a novel protocol (foldGPCR) for modeling the transmembrane (TM) region of GPCRs in complex with a ligand, aimed to accurately model the structural divergence between the template and target in the TM helices. The protocol is based on predicted conserved inter-residue contacts between the template and target, and exploits an all-atom implicit membrane force field. The placement of the ligand in the binding pocket is guided by biochemical data. The foldGPCR protocol is implemented by a stepwise hierarchical approach, in which the TM helical bundle and the ligand are assembled by simulated annealing trials in the first step, and the receptor-ligand complex is refined with replica exchange sampling in the second step. The protocol is applied to model the human beta(2)-adrenergic receptor (beta(2)AR) bound to carazolol, using contacts derived from the template structure of bovine rhodopsin. Comparison with the X-ray crystal structure of the beta(2)AR shows that our protocol is particularly successful in accurately capturing helix backbone irregularities and helix-helix packing interactions that distinguish rhodopsin from beta(2)AR.