Solution structure of the second extracellular loop of human thromboxane A2 receptor

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.     

PREDICT modeling and in-silico screening for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are a major group of drug targets for which only one x-ray structure is known (the nondrugable rhodopsin), limiting the application of structure-based drug discovery to GPCRs. In this paper we present the details of PREDICT, a new algorithmic approach for modeling the 3D structure of GPCRs without relying on homology to rhodopsin. PREDICT, which focuses on the transmembrane domain of GPCRs, starts from the primary sequence of the receptor, simultaneously optimizing multiple 'decoy' conformations of the protein in order to find its most stable structure, culminating in a virtual receptor-ligand complex. In this paper we present a comprehensive analysis of three PREDICT models for the dopamine D2, neurokinin NK1, and neuropeptide Y Y1 receptors. A shorter discussion of the CCR3 receptor model is also included. All models were found to be in good agreement with a large body of experimental data. The quality of the PREDICT models, at least for drug discovery purposes, was evaluated by their successful utilization in in-silico screening. Virtual screening using all three PREDICT models yielded enrichment factors 9-fold to 44-fold better than random screening. Namely, the PREDICT models can be used to identify active small-molecule ligands embedded in large compound libraries with an efficiency comparable to that obtained using crystal structures for non-GPCR targets.     

Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively.     

Selective reconstitution of human D4 dopamine receptor variants with Gi alpha subtypes

G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.     

Protein‐based virtual screening of chemical databases. II. Are homology models of g‐protein coupled receptors suitable targets?

The aim of the current study is to investigate whether homology models of G-Protein-Coupled Receptors (GPCRs) that are based on bovine rhodopsin are reliable enough to be used for virtual screening of chemical databases. Starting from the recently described 2.8 A-resolution X-ray structure of bovine rhodopsin, homology models of an "antagonist-bound" form of three human GPCRs (dopamine D3 receptor, muscarinic M1 receptor, vasopressin V1a receptor) were constructed. The homology models were used to screen three-dimensional databases using three different docking programs (Dock, FlexX, Gold) in combination with seven scoring functions (ChemScore, Dock, FlexX, Fresno, Gold, Pmf, Score). Rhodopsin-based homology models turned out to be suitable, indeed, for virtual screening since known antagonists seeded in the test databases could be distinguished from randomly chosen molecules. However, such models are not accurate enough for retrieving known agonists. To generate receptor models better suited for agonist screening, we developed a new knowledge- and pharmacophore-based modeling procedure that might partly simulate the conformational changes occurring in the active site during receptor activation. Receptor coordinates generated by this new procedure are now suitable for agonist screening. We thus propose two alternative strategies for the virtual screening of GPCR ligands, relying on a different set of receptor coordinates (antagonist-bound and agonist-bound states).     

Sequential Application of Ligand and Structure Based Modeling Approaches to Index Chemicals for Their hH4R Antagonism

The human histamine H₄ receptor (hH₄R), a member of the G-protein coupled receptors (GPCR) family, is an increasingly attractive drug target. It plays a key role in many cell pathways and many hH₄R ligands are studied for the treatment of several inflammatory, allergic and autoimmune disorders, as well as for analgesic activity. Due to the challenging difficulties in the experimental elucidation of hH₄R structure, virtual screening campaigns are normally run on homology based models. However, a wealth of information about the chemical properties of GPCR ligands has also accumulated over the last few years and an appropriate combination of these ligand-based knowledge with structure-based molecular modeling studies emerges as a promising strategy for computer-assisted drug design. Here, two chemoinformatics techniques, the Intelligent Learning Engine (ILE) and Iterative Stochastic Elimination (ISE) approach, were used to index chemicals for their hH₄R bioactivity. An application of the prediction model on external test set composed of more than 160 hH₄R antagonists picked from the chEMBL database gave enrichment factor of 16.4. A virtual high throughput screening on ZINC database was carried out, picking ∼4000 chemicals highly indexed as H₄R antagonists'' candidates. Next, a series of 3D models of hH₄R were generated by molecular modeling and molecular dynamics simulations performed in fully atomistic lipid membranes. The efficacy of the hH₄R 3D models in discrimination between actives and non-actives were checked and the 3D model with the best performance was chosen for further docking studies performed on the focused library. The output of these docking studies was a consensus library of 11 highly active scored drug candidates. Our findings suggest that a sequential combination of ligand-based chemoinformatics approaches with structure-based ones has the potential to improve the success rate in discovering new biologically active GPCR drugs and increase the enrichment factors in a synergistic manner.     

The extreme C-terminal region of Gαs differentially couples to the luteinizing hormone and beta2-adrenergic receptors

The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein α-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of Gα(s) is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and β(2)-adrenergic receptor (β(2)-AR), a model cell system was created for the expression and manipulation of Gα(s) subunits in LHR(+) s49 ck cells that lack endogenous Gα(s). On the basis of studies involving truncations, mutations, and chain extensions of Gα(s), the CT was found to be necessary for LHR and β(2)-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the Gα(s) CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of Gα(s) with residues in LHR and β(2)-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of Gα(s) recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of β(2)-AR are expected to contribute significantly to differences in Gα(s) recognition by the two receptors.     

Modeling and docking of the three-dimensional structure of the human melanocortin 4 receptor

A three-dimensional structure of the human melanocortin 4 receptor (hMC4R) is constructed in this study using a computer-aided molecular modeling approach. Human melanocortin 4 receptor is a G Protein-Coupled Receptor (GPCR). We structurally aligned transmembrane helices with bovine rhodopsin transmembrane domains, simulated both intracellular and extracellular loop domains on homologous loop regions in other proteins of known 3D structure and modeled the C terminus on the corresponding part of bovine rhodopsin. Then tandem minimization and dynamics calculations were run to refine the crude structure. The simulative model was tested by docking with a triplet peptide (RFF) ligand. It was found that the ligand is located among transmembrane regions TM3, TM4, TM5, and TM6 of hMC4R. In consistence with mutational and biochemical data, binding site is mainly formed as a hydrophobic and negatively charged pocket. The model constructed here might provide a structural framework for making rational predictions in relevant fields.     

3D modeling of the activated states of constitutively active mutants of rhodopsin

The activated (R*) states in constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) are presumably characterized by lower energies than the resting (R) states. If specific configurations of TM helices differing by rotations along the long transmembrane axes possess energies lower than that in the R state for pronounced CAMs, but not for non-CAMs, these particular configurations of TM helices are candidate 3D models for the R* state. The hypothesis was studied in the case of rhodopsin, the only GPCR for which experimentally determined 3D models of the R and R* states are currently available. Indeed, relative energies of the R* state were significantly lower than that of the R state for the rhodopsin mutants G90D/M257Y and E113Q/M257Y (strong CAMs), but not for G90D, E113Q, and M257Y (not CAMs). Next, the developed build-up procedure successfully identified few similar configurations of the TM helical bundle of G90D/M257Y and E113Q/M257Y as possible candidates for the 3D model of the R* state of rhodopsin, all of them being in good agreement with the model suggested by experiment. Since constitutively active mutants are known for many of GPCRs belonging to the large rhodopsin-like family, this approach provides a way for predicting possible 3D structures corresponding to the activated states of the TM regions of many GPCRs for which CAMs have been identified.     

Receptor-antagonist interactions in the complexes of agouti and agouti-related protein with human melanocortin 1 and 4 receptors

The molecular interactions between human melanocortin receptor-1 and -4 (hMC1R and hMC4R) and their endogenous antagonists, agouti signaling protein (ASIP) and agouti-related protein (AGRP), were assessed by studying the effects of site-directed mutations on the binding affinity of (125)I-ASIP[90-132(L89Y)] and (125)I-AGRP(86-132). Mutations of homologous residues from transmembrane helices (TMHs) 3 and 6 and extracellular loop (EL) 3 (D121A, T124A, F257A, and F277M in hMC1R and D126A, I129A F261A, and M281F in hMC4R) impaired binding of both antagonists to hMC4R and binding of the ASIP fragment to hMC1R. However, the mutations in TMH2 (E94A in hMC1R and E100A in hMC4R), TMH7 (F280A in hMC1R and F284A in hMC4R), and EL2 (Y183S, H184S, and D184H in hMC1R) only significantly affected binding of the ASIP fragment. The dependence of agonist binding on the dithiothreitol concentration followed a monophasic curve for wild-type hMC4R and its C40A, C271A, and C279A mutants and a biphasic curve for hMC1R, suggesting the presence of at least one structurally and functionally essential disulfide bond in both wild-type receptors and the hMC4R mutants. Models of complexes of both receptors with the ASIP fragment and hMC4R with the AGRP fragment were calculated using constraints from the experimental structures of rhodopsin and AGRP fragments, a set of deduced hydrogen bonds, supplemented by two proposed disulfide bridges and receptor-ligand contacts, derived from our mutagenesis data. In the models of the ASIP fragment complexed with both receptors, the core ligand tripeptide, Arg-Phe-Phe, positioned between TMHs 3 and 6, is shifted toward TMHs 2 and 7 relative to its position in the AGRP-hMC4R model, while the N-terminal loop and two central disulfides of the antagonists interact with EL2 of the receptors.     

Structure-based identification of binding sites,native ligands and potential inhibitors for G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest family of cell-surface receptors involved in signal transmission. Drugs associated with GPCRs represent more than one fourth of the 100 top-selling drugs and are the targets of more than half of the current therapeutic agents on the market. Our methodology based on the internal coordinate mechanics (ICM) program can accurately identify the ligand-binding pocket in the currently available crystal structures of seven transmembrane (7TM) proteins [bacteriorhodopsin (BR) and bovine rhodopsin (bRho)]. The binding geometry of the ligand can be accurately predicted by ICM flexible docking with and without the loop regions, a useful finding for GPCR docking because the transmembrane regions are easier to model. We also demonstrate that the native ligand can be identified by flexible docking and scoring in 1.5% and 0.2% (for bRho and BR, respectively) of the best scoring compounds from two different types of compound database. The same procedure can be applied to the database of available chemicals to identify specific GPCR binders. Finally, we demonstrate that even if the sidechain positions in the bRho binding pocket are entirely wrong, their correct conformation can be fully restored with high accuracy (0.28 A) through the ICM global optimization with and without the ligand present. These binding site adjustments are critical for flexible docking of new ligands to known structures or for docking to GPCR homology models. The ICM docking method has the potential to be used to "de-orphanize" orphan GPCRs (oGPCRs) and to identify antagonists-agonists for GPCRs if an accurate model (experimentally and computationally validated) of the structure has been constructed or when future crystal structures are determined.     

Towards an understanding of the structural basis for insect olfaction by odorant receptors

Insects have co-opted a unique family of seven transmembrane proteins for odour sensing. Odorant receptors are believed to have evolved from gustatory receptors somewhere at the base of the Hexapoda and have expanded substantially to become the dominant class of odour recognition elements within the Insecta. These odorant receptors comprise an obligate co-receptor, Orco, and one of a family of highly divergent odorant “tuning” receptors. The two subunits are thought to come together at some as-yet unknown stoichiometry to form a functional complex that is capable of both ionotropic and metabotropic signalling. While there are still no 3D structures for these proteins, site-directed mutagenesis, resonance energy transfer, and structural modelling efforts, all mainly on Drosophila odorant receptors, are beginning to inform hypotheses of their structures and how such complexes function in odour detection. Some of the loops, especially the second extracellular loop that has been suggested to form a lid over the binding pocket, and the extracellular regions of some transmembrane helices, especially the third and to a less extent the sixth and seventh, have been implicated in ligand recognition in tuning receptors. The possible interaction between Orco and tuning receptor subunits through the final intracellular loop and the adjacent transmembrane helices is thought to be important for transducing ligand binding into receptor activation. Potential phosphorylation sites and a calmodulin binding site in the second intracellular loop of Orco are also thought to be involved in regulating channel gating. A number of new methods have recently been developed to express and purify insect odorant receptor subunits in recombinant expression systems. These approaches are enabling high throughput screening of receptors for agonists and antagonists in cell-based formats, as well as producing protein for the application of biophysical methods to resolve the 3D structure of the subunits and their complexes.     

Structure-function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Structure–function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Similar structures and shared switch mechanisms of the beta2-adrenoceptor and the parathyroid hormone receptor. Zn(II) bridges between helices III and VI block activation

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A. , Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the beta2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.     

The neural γ<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript> gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ₂α₁β₂α₁β₂ gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ₂α₁β₂α₁β₂ has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ₂ and 2 α₁ subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models—one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2–M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.     

Preliminary mutational analysis of the human kinin B2 receptor for nonpeptide antagonist ligands recognition

FR173657, LF16,0335, and LF16,0687 are nonpeptide antagonists, endowed with high affinity and selectivity for the human kinin B2 receptor. The kinin B2 receptor belongs to the family of G-protein-coupled receptors with seven transmembrane (TM) helices. In the present study, we aimed, through computer-assisted modeling and mutagenesis, to identify residues in the human B2 receptor (hB2R) amino acid sequence that are involved in nonpeptide antagonist binding in order to build up experimental data as a first step towards a molecular model of nonpeptide ligands binding site. Fourteen amino acid residues within the TM segments were mutated to alanine. The wild type and mutant receptors were stably expressed in Chinese hamster ovary (dhfr-) cells and tested for their ability to bind agonist ([3H]bradykinin) and peptide antagonist ([3H]MENI 1270) radioligands. The affinity of nonpeptide ligands was determined by heterologous competition experiments using the above radioligands. We found that some mutations in TM2 (W86A) and TM7 (Y295A, N297A) impair the binding affinity of the three nonpeptide antagonists. On the other hand, some mutated residues in TM3 (S1 17A) and TM6 (W256A) reduce the affinity of LF16,0335 and LF16,0687 only. Results are discussed in order to build up a hypothesis for the likely different interactions of various nonpeptide ligands with the B2 receptor.     

Modeling G protein‐coupled receptors for structure‐based drug discovery using low‐frequency normal modes for refinement of homology models: Application to H3 antagonists

G Protein‐Coupled Receptors (GPCRs) are integral membrane proteins that play important role in regulating key physiological functions, and are targets of about 50% of all recently launched drugs. High‐resolution experimental structures are available only for very few GPCRs. As a result, structure‐based drug design efforts for GPCRs continue to rely on in silico modeling, which is considered to be an extremely difficult task especially for these receptors. Here, we describe Gmodel, a novel approach for building 3D atomic models of GPCRs using a normal mode‐based refinement of homology models. Gmodel uses a small set of relevant low‐frequency vibrational modes derived from Random Elastic Network model to efficiently sample the large‐scale receptor conformation changes and generate an ensemble of alternative models. These are used to assemble receptor–ligand complexes by docking a known active into each of the alternative models. Each of these is next filtered using restraints derived from known mutation and binding affinity data and is refined in the presence of the active ligand. In this study, Gmodel was applied to generate models of the antagonist form of histamine 3 (H3) receptor. The validity of this novel modeling approach is demonstrated by performing virtual screening (using the refined models) that consistently produces highly enriched hit lists. The models are further validated by analyzing the available SAR related to classical H3 antagonists, and are found to be in good agreement with the available experimental data, thus providing novel insights into the receptor–ligand interactions. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

The dopamine D4 receptor,the ultimate disordered protein

The human D4 dopamine receptor is a synaptic neurotransmitter receptor responsible for neuronal signaling in the mesolimbic system of the brain, an area of the brain that regulates emotion and complex behavior. Its structure makes it a very unusual and interesting G protein-coupled receptor (GPCR) as it has several polymorphic variants of its gene in the region encoding the third intracellular loop (IL3). This region contains from two to seven or more similar 48 base pair repeats. These repeats cause this protein to have a very high disorder index and this, in turn, makes it very interactive with other proteins. Among GPCRs in general, the unusually proline-rich IL3 is unique to the D4 receptor (D4R). We believe that, as in the D2R, this region of the receptor plays a role in it’s interaction with other receptors.