Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.     

Genetic analysis of the first and third extracellular loops of the C5a receptor reveals an essential WXFG motif in the first loop

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.     

Role of the extracellular loops of G protein-coupled receptors in ligand recognition: a molecular modeling study of the human P2Y1 receptor

The P2Y1 receptor is a G protein-coupled receptor (GPCR) and is stimulated by extracellular ADP and ATP. Site-directed mutagenesis of the three extracellular loops (ELs) of the human P2Y1 receptor indicates the existence of two essential disulfide bridges (Cys124 in EL1 and Cys202 in EL2; Cys42 in the N-terminal segment and Cys296 in EL3) and several specific ionic and H-bonding interactions (involving Glu209 and Arg287). Through molecular modeling and molecular dynamics simulations, an energetically sound conformational hypothesis for the receptor has been calculated that includes transmembrane (TM) domains (using the electron density map of rhodopsin as a template), extracellular loops, and a truncated N-terminal region. ATP may be docked in the receptor, both within the previously defined TM cleft and within two other regions of the receptor, termed meta-binding sites, defined by the extracellular loops. The first meta-binding site is located outside of the TM bundle, between EL2 and EL3, and the second higher energy site is positioned immediately underneath EL2. Binding at both the principal TM binding site and the lower energy meta-binding sites potentially affects the observed ligand potency. In meta-binding site I, the side chain of Glu209 (EL2) is within hydrogen-bonding distance (2.8 A) of the ribose O3', and Arg287 (EL3) coordinates both alpha- and beta-phosphates of the triphosphate chain, consistent with the insensitivity in potency of the 5'-monophosphate agonist, HT-AMP, to mutation of Arg287 to Lys. Moreover, the selective reduction in potency of 3'NH2-ATP in activating the E209R mutant receptor is consistent with the hypothesis of direct contact between EL2 and nucleotide ligands. Our findings support ATP binding to at least two distinct domains of the P2Y1 receptor, both outside and within the TM core. The two disulfide bridges present in the human P2Y1 receptor play a major role in the structure and stability of the receptor, to constrain the loops within the receptor, specifically stretching the EL2 over the opening of the TM cleft and thus defining the path of access to the binding site.     

NMR structure of the thromboxane A2 receptor ligand recognition pocket.

To overcome the difficulty of characterizing the structures of the extracellular loops (eLPs) of G protein-coupled receptors (GPCRs) other than rhodopsin, we have explored a strategy to generate a three-dimensional structural model for a GPCR, the thromboxane A(2) receptor. This three-dimensional structure was completed by the assembly of the NMR structures of the computation-guided constrained peptides that mimicked the extracellular loops and connected to the conserved seven transmembrane domains. The NMR structure-based model reveals the structural features of the eLPs, in which the second extracellular loop (eLP(2)) and the disulfide bond between the first extracellular loop (eLP(1)) and eLP(2) play a major role in forming the ligand recognition pocket. The eLP(2) conformation is dynamic and regulated by the oxidation and reduction of the disulfide bond, which affects ligand docking in the initial recognition. The reduced form of the thromboxane A(2) receptor experienced a decrease in ligand binding activity due to the rearrangement of the eLP(2) conformation. The ligand-bound receptor was, however, resistant to the reduction inactivation because the ligand covered the disulfide bond and stabilized the eLP(2) conformation. This molecular mechanism of ligand recognition is the first that may be applied to other prostanoid receptors and other GPCRs.     

Plasticity of the ligand binding pocket in the bitter taste receptor T2R7

Bitter taste receptors (T2Rs) are a group of 25 G protein-coupled receptors (GPCRs) in humans. The cognate agonists and the mechanism of ligand binding to the majority of the T2Rs remain unknown. Here we report the first structure-function analysis of T2R7 and study the ability of this receptor to bind to different agonists by site-directed mutagenesis. Screening of ligands for T2R7 in calcium based assays lead to the identification of novel compounds that activate this receptor. Quinine, diphenidol, dextromethorphan and diphenhydramine showed substantial activation of T2R7. Interestingly, these bitter compounds showed different pharmacological characteristics. To investigate the structural features in T2R7 that might contribute to the observed differences in agonist specificities, molecular model guided ligand docking and site-directed mutagenesis was pursued. Amino acids D65, D86, W89, N167, T169, W170, S181, T255 and E271 in the ligand-binding pocket were replaced and the mutants characterized pharmacologically. Our results suggest D86, S181 and W170 present on the extracellular side of transmembrane 3 (TM3), TM5 and in extracellular loop 2 (ECL2) are essential for agonist binding in T2R7. Mutations of these amino acids lead to loss-of-function. We also identified gain-of-function residues that are agonist specific. These results suggest that agonists bind at an extracellular site rather than deep within the TM core involving residues present in both ECL2 and TM helices in T2R7. Similar to majority of the Class A GPCRs, ECL2 in T2R7 plays a significant role in agonist binding and activation.     

Structural insights into the role of the ACTH receptor cysteine residues on receptor function

The ACTH receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release. Human MC2R (hMC2R) has 10 cysteine residues, which are located in extracellular loops (ELs), transmembrane domains (TMs), and intracellular loops (ILs). In this study, we examined the importance of these cysteine residues in receptor function and determined their involvement in disulfide bond formation. We replaced these cysteines with serine and expressed the mutated receptors in adrenal OS3 cells, which lack endogenous MC2R. Our results indicate that four mutations, C21S in NH(2) terminus, C245S, C251S, and C253S in EL3, resulted in significant decrease both in receptor expression and receptor function. Mutation of cysteine 231 in TM6 significantly decreased ACTH binding affinity and potency. In contrast, the five other mutated receptors (C64S, C158S, C191S, C267S, and C293S) did not significantly alter ACTH binding affinity and potency. These results suggest that extracellular cysteine residue 21, 245, 251, and 253, as well as transmembrane cysteine residue 231 are crucial for ACTH binding and signaling. Further experiments suggest that a disulfide bond exists between the residue C245 and C251 in EL3. These findings provide important insights into the importance of cysteine residues of hMC2R for receptor function.     

Role of extracellular domains in PBAN/pyrokinin GPCRs from insects using chimera receptors

Pheromone biosynthesis-activating neuropeptide (PBAN) is a peptide used by a variety of moths to regulate pheromone production. Pyrokinins are peptides that activate muscle contraction in a variety of insects. These peptides have a common FXPRLamide C-terminal ending that is required for activity. Receptors have been identified from a moth and Drosophila as belonging to the rhodopsin family of G-protein coupled receptors (GPCRs) with sequence similarity to neuromedin U receptors from vertebrates. No insect GPCR has been characterized with regard to role of extracellular domains required for peptide binding and receptor activation. To begin characterizing these GPCRs we created chimera receptors using a PBAN-receptor from a moth and pyrokinin-receptors from Drosophila where extracellular domains were swapped. The N-terminal of the moth GPCR has two N-glycosylation sites that when replaced with glutamines, activity was reduced but not absent, indicating these sites contribute to receptor stability. Activity was greatly reduced by replacing the 2nd extracellular loop that has an N-glycosylation site and a cysteine that can form a disulfide bridge with a cysteine at the beginning of the 3rd transmembrane domain. Exchange of the 3rd extracellular loop between the moth and Drosophila receptor resulted in differential activation by PBAN or a diapause hormone peptide. This result indicates that the 3rd extracellular loop is directly involved in peptide ligand recognition. Results are discussed in context of the structural features of insect GPCRs that are required for receptor activation as compared to vertebrate receptors.     

A unique hormonal recognition feature of the human glucagon-like peptide-2 receptor

Glucagon-like peptides (GLP-1 and GLP-2) are two proglucagon-derived intestinal hormones that mediate distinct physiological functions through two related receptors (GLP-1R and GLP-2R) which are important drug targets for metabolic disorders and Crohn’s disease, respectively. Despite great progress in GLP-1R structure determination, our understanding on the differences of peptide binding and signal transduction between these two receptors remains elusive. Here we report the electron microscopy structure of the human GLP-2R in complex with GLP-2 and a G_s heterotrimer. To accommodate GLP-2 rather than GLP-1, GLP-2R fine-tunes the conformations of the extracellular parts of transmembrane helices (TMs) 1, 5, 7 and extracellular loop 1 (ECL1). In contrast to GLP-1, the N-terminal histidine of GLP-2 penetrates into the receptor core with a unique orientation. The middle region of GLP-2 engages with TM1 and TM7 more extensively than with ECL2, and the GLP-2 C-terminus closely attaches to ECL1, which is the most protruded among 9 class B G protein-coupled receptors (GPCRs). Functional studies revealed that the above three segments of GLP-2 are essential for GLP-2 recognition and receptor activation, especially the middle region. These results provide new insights into the molecular basis of ligand specificity in class B GPCRs and may facilitate the development of more specific therapeutics.Subject terms: Cryoelectron microscopy, Hormone receptors     

Active peptidic mimics of the second intracellular loop of the V(1A) vasopressin receptor are structurally related to the second intracellular rhodopsin loop: a combined 1H NMR and biochemical study

Vasopressin (VP) receptors belong to the widespread G protein-coupled receptor family. The crucial role of VP receptor intracellular loops in the coupling with the heterotrimeric G proteins was previously demonstrated by construction of a vasopressin receptor chimera. Yet, no fine structural data are available concerning the receptor molecular determinants involved in their interactions with G proteins. In this study, we synthesized both a linear and a cyclic form of the second intracellular loop (i2) of the human V(1a) vasopressin receptor isoform that is important for the interaction between the alphaq/alpha11 G protein and the receptor. These two peptides are biologically active. They specifically inhibit vasopressin binding to the V(1a) receptor, suggesting that the corresponding endogenous peptides contribute to the structure of the vasopressin binding site via intra- or intermolecular interactions with the core of the V(1a) receptor. The i2 peptide structures were determined by (1)H NMR. Both exhibit a helix and helical elements in their N- and C-terminal parts, respectively, separated by a turn imposed by a proline residue. More interestingly, the central Pro-Leu motif conserved in many GPCRs and thought to be important for coupling to G proteins can adopt different conformations. The "U" shape structure of the i2 loop is compatible with its anchoring to transmembrane domains III and IV and is very similar to the shape of bovine rhodopsin i2. Altogether, these data contribute to a better understanding of the structure of a not yet crystallized GPCR using the mimetic peptide approach.     

Extracellular domains of the neurokinin-1 receptor: structural characterization and interactions with substance P

The technical difficulties associated with the structure determination of membrane proteins have limited the structural information available for the ligand binding to G-protein coupled receptors (GPCRs). Here, we describe a reductionist approach to GPCR structure determination in which the extracellular domains of the receptor are examined by high-resolution NMR in the presence of a membrane mimetic. The resulting structural features are then incorporated into a molecular model of the receptor, utilizing the x-ray structure of rhodopsin to generate the topological orientation of the transmembrane helices. The results of our study of the neurokinin-1 receptor (NK-1R) and its interactions with substance P (SP) are detailed here. The structure of the N-terminus, NK-1R(1-39), and of the third extracellular loop, NK-1R(264-290), in the presence of dodecylphosphocholine micelles is described. Our findings provide a structural basis for the interpretation of the results from other methods including mutagenesis, fluorescence, and photoaffinity labeling experiments, resulting in an experimentally based, high-resolution model of SP binding to NK-1R.     

Structural implication for receptor oligomerization from functional reconstitution studies of mutant V2 vasopressin receptors

Previous studies have established that G-protein-coupled receptors (GPCRs) are composed of independent folding domains. Based on this findings we attempted to rescue the function of clinically relevant missense mutations (R137H, S167L, and R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R), by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptor constructs in COS-7 cells. Coimmunoprecipitation and enzyme-linked immunosorbent assay studies demonstrated a specific association of E242X with full-length V2-Rs even in the presence of missense mutations. Systematic analysis of the structural requirements for the observed receptor/fragment association showed that N-terminal fragments containing at least transmembrane regions 1-3 interact with the full-length V2-R. Despite this specific interaction, no functional reconstitution was achieved for mutant V2-Rs following coexpression with E242X and Y280C. However, functional activity of R137H and R181C upon coexpression with E242X was regained by mutational disruption of the extracellular disulfide bond, which is highly conserved among GPCRs. Our data with the V2-R are consistent with a structural model in which class I GPCRs form contact oligomers by lateral interaction rather than by a domain-swapping mechanism.     

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.     

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.     

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASM^CXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASM^CXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASM^CXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.     

Structural aspects of M? muscarinic acetylcholine receptor dimer formation and activation

To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (～50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.     

Structural mechanisms of constitutive activation in the C5a receptors with mutations in the extracellular loops: molecular modeling study

Previously we demonstrated by random saturation mutagenesis a set of mutations in the extracellular (EC) loops that constitutively activate the C5a receptor (C5aR) (Klco et al., Nat Struct Mol Biol 2005;12:320-326; Klco et al., J Biol Chem 2006;281:12010-12019). In this study, molecular modeling revealed possible conformations for the extracellular loops of the C5a receptors with mutations in the EC2 loop or in the EC3 loop. Comparison of low-energy conformations of the EC loops defined two distinct clusters of conformations typical either for strongly constitutively active mutants of C5aR (the CAM cluster) or for nonconstitutively active mutants (the non-CAM cluster). In the CAM cluster, the EC3 loop was turned towards the transmembrane (TM) helical bundle and more closely interacted with EC2 than in the non-CAM cluster. This suggested a structural mechanism of constitutive activity where EC3 contacts EC2 leading to EC2 interactions with helix TM3, thus triggering movement of TM7 towards TM2 and TM3. The movement initiates rearrangement of the system of hydrogen bonds between TM2, TM3 and TM7 including formation of the hydrogen bond between the side chains of D82(2.50) in TM2 and N296(7.49) in TM7, which is crucial for formation of the activated states of the C5a receptors (Nikiforovich et al., Proteins: Struct Funct Gene 2011;79:787-802). Since the relative large length of EC3 in C5aR (13 residues) is comparable with those in many other members of rhodopsin family of GPCRs (13-19 residues), our findings might reflect general mechanisms of receptor constitutive activation. The very recent X-ray structure of the agonist-induced constitutively active mutant of rhodopsin (Standfuss et al., Nature 2011;471:656-660) is discussed in view of our modeling results.     

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

The extracellular domains of the thromboxane A2 receptor (TP receptor) were found to be involved in the specific ligand recognition. Determination of the three-dimensional (3D) structure of the extracellular loops would help to explain the mechanism of the ligand binding to its receptor with regard to the tertiary structure. Based on our previous studies on the extracellular loop of the human TP receptor, the synthetic loop peptides, whose termini are constrained to 10 to 14-A separations, are more likely to mimic the native structure of the extracellular loops. In this study, a peptide with the sequence of the third extracellular loop (eLP3, residues 271-289) of the TP receptor was synthesized, and its termini were constrained by the formation of a disulfide bond between the additional homocysteines located at both ends. Fluorescence spectroscopic studies showed that the fluorescence intensity of this constrained loop peptide could be increased by the addition of SQ29,548, a TP receptor antagonist, which indicated the interaction between the peptide and the ligand. The structure of this peptide was then studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. 1H NMR assignments of the peptide were obtained and structure constraints were derived from nuclear Overhauser effects and J-coupling constants. The solution structure of the peptide was then calculated based on these constraints. The overall structure shows a beta turn from residues 278 to 281. It also shows a distance of 9.45A between the ends of the N and C termini of the peptide, which agrees with the distance between the two residues at the ends of the transmembrane helices connecting the eLP3 on the TP receptor working model generated using molecular modeling, based on the crystal structure of bovine rhodopsin. These results provide valuable information for the characterization of the complete 3D structure of the extracellular domains of the human TP receptor.     

Molecular characterization of the substance P*neurokinin-1 receptor complex: development of an experimentally based model

Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed. The ligand.receptor complex is based on experimental data from a series of photoaffinity labeling experiments and spectroscopic structural studies of extracellular domains of the NK-1R. Using the ligand/receptor contact points derived from incorporation of photolabile probes (p-benzoylphenylalanine (Bpa)) into SP at positions 3, 4, and 8 and molecular dynamics simulations, the topological arrangement of SP within the NK-1R is explored. The model incorporates the structural features, determined by high resolution NMR studies, of the second extracellular loop (EC2), containing contact points Met(174) and Met(181), providing important experimentally based conformational preferences for the simulations. Extensive molecular dynamics simulations were carried out to probe the nature of the two contact points identified for the Bpa(3)SP analogue (Bremer, A. A., Leeman, S. E., and Boyd, N. D. (2001) J. Biol. Chem. 276, 22857-22861), examining modes of ligand binding in which the contact points are fulfilled sequentially or simultaneously. The resulting ligand.receptor complex has the N terminus of SP projecting toward transmembrane helix (TM) 1 and TM2, exposed to the solvent. The C terminus of SP is located in proximity to TM5 and TM6, deeper into the central core of the receptor. The central portion of the ligand, adopting a helical loop conformation, is found to align with the helices of the central regions EC2 and EC3, forming important interactions with both of these extracellular domains. The model developed here allows for atomic insight into the biochemical data currently available and guides targeting of future experiments to probe specific ligand/receptor interactions and thereby furthers our understanding of the functioning of this important neuropeptide system.