Crystal Structure Of Bovine Rhodopsin Research Articles

Detection of Pairwise Residue Proximity by Covariation Analysis for 3D-Structure Prediction of G-Protein-Coupled Receptors

G-protein-coupled receptor (GPCR) is one of the most important targets for medicines. Homology modeling based on the crystal structure of bovine rhodopsin is currently the most frequently used method for GPCR targeted drug design. Information about residue-residue contacts and the structural specificity in the subfamily is essential for constructing more precise 3D structures, to distinguish the structural differences between the template and targets. In this study, we adopted the covariation analysis to extract information about residue-residue interactions from the amino acid sequence. In the opsin family, a large number of adjacent covarying residue pairs were detected. The detected residue pairs have a strong tendency to gather in some regions important for the structure and function. These results suggest that the covariation analysis is practically utilized to detect adjacent residue pairs and also to apply for predicting functional sites. Analyses of other GPCR subfamilies, olfactory receptor and chemokine receptor families, demonstrated that some adjacent covarying residue pairs were common. Thus, the covariation analysis has possibilities in the substantial improvement of the 3D-structure modeling of GPCRs and in the detection of functional sites such as the ligand-binding sites.

Binding site analysis of full-length α1a adrenergic receptor using homology modeling and molecular docking

The recent availability of crystal structure of bovine rhodopsin offers new opportunities in order to approach the construction of G protein coupled receptors. This study focuses the attention on the modeling of full-length α1a adrenergic receptor (α1a-AR) due to its biological role and significant implications in pharmacological treatment of benign prostate hyperplasia. This work could be considered made up by two main steps: (a) the construction of full structure of α1a-AR, through homology modeling methods; (b) the automated docking of an endogenous agonist, norepinephrine, and of an antagonist, WB-4101, using BioDock program. The obtained results highlight the key residues involved in binding sites of both agonists and antagonists, confirming the mutagenesis data and giving new suggestions for the rational design of selective ligands.

Structural studies of the putative helix 8 in the human $beta;2 adrenergic receptor: an NMR study

The recently reported crystal structure of bovine rhodopsin revealed a cytoplasmic helix (helix 8) in addition to the seven transmembrane helices. This domain is roughly perpendicular to the transmembrane bundle in the presence of an interface and may be a loop-like structure in the absence of an interface. Several studies carried out on this domain suggested that it might act as a conformational switch between the inactive and activated states of this G-protein coupled receptor (GPCR). These results raised the question whether helix 8 may be an important feature of other GPCRs as well. To explore this question, we determined the structure of a peptide representing the putative helix 8 domain in another receptor that belongs to the rhodopsin family of GPCRs, the human β 2 adrenergic receptor (hβ 2 AR), using two-dimensional 1 H nuclear magnetic resonance (NMR). The key results from this structural study are that the putative helix 8 domain is helical in detergent and in DMSO while in water this region is disordered; the conformation is therefore dependent upon the environment. Comparison of data from five GPCRs suggests that these observations may be generally important for GPCR structure and function.

Structural studies of the putative helix 8 in the human β 2 adrenergic receptor: an NMR study

The recently reported crystal structure of bovine rhodopsin revealed a cytoplasmic helix (helix 8) in addition to the seven transmembrane helices. This domain is roughly perpendicular to the transmembrane bundle in the presence of an interface and may be a loop-like structure in the absence of an interface. Several studies carried out on this domain suggested that it might act as a conformational switch between the inactive and activated states of this G-protein coupled receptor (GPCR). These results raised the question whether helix 8 may be an important feature of other GPCRs as well. To explore this question, we determined the structure of a peptide representing the putative helix 8 domain in another receptor that belongs to the rhodopsin family of GPCRs, the human β 2 adrenergic receptor (hβ 2AR), using two-dimensional 1H nuclear magnetic resonance (NMR). The key results from this structural study are that the putative helix 8 domain is helical in detergent and in DMSO while in water this region is disordered; the conformation is therefore dependent upon the environment. Comparison of data from five GPCRs suggests that these observations may be generally important for GPCR structure and function.

First Principles Predictions of the Structure and Function of G-Protein-Coupled Receptors: Validation for Bovine Rhodopsin

G-protein-coupled receptors (GPCRs) are involved in cell communication processes and with mediating such senses as vision, smell, taste, and pain. They constitute a prominent superfamily of drug targets, but an atomic-level structure is available for only one GPCR, bovine rhodopsin, making it difficult to use structure-based methods to design receptor-specific drugs. We have developed the MembStruk first principles computational method for predicting the three-dimensional structure of GPCRs. In this article we validate the MembStruk procedure by comparing its predictions with the high-resolution crystal structure of bovine rhodopsin. The crystal structure of bovine rhodopsin has the second extracellular (EC-II) loop closed over the transmembrane regions by making a disulfide linkage between Cys-110 and Cys-187, but we speculate that opening this loop may play a role in the activation process of the receptor through the cysteine linkage with helix 3. Consequently we predicted two structures for bovine rhodopsin from the primary sequence (with no input from the crystal structure)—one with the EC-II loop closed as in the crystal structure, and the other with the EC-II loop open. The MembStruk-predicted structure of bovine rhodopsin with the closed EC-II loop deviates from the crystal by 2.84 Å coordinate root mean-square (CRMS) in the transmembrane region main-chain atoms. The predicted three-dimensional structures for other GPCRs can be validated only by predicting binding sites and energies for various ligands. For such predictions we developed the HierDock first principles computational method. We validate HierDock by predicting the binding site of 11- cis-retinal in the crystal structure of bovine rhodopsin. Scanning the whole protein without using any prior knowledge of the binding site, we find that the best scoring conformation in rhodopsin is 1.1 Å CRMS from the crystal structure for the ligand atoms. This predicted conformation has the carbonyl O only 2.82 Å from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 0.62 Å CRMS from the crystal structure. We also used HierDock to predict the binding site of 11- cis-retinal in the MembStruk-predicted structure of bovine rhodopsin (closed loop). Scanning the whole protein structure leads to a structure in which the carbonyl O is only 2.85 Å from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 2.92 Å CRMS from the crystal structure. The good agreement of the ab initio-predicted protein structures and ligand binding site with experiment validates the use of the MembStruk and HierDock first principles’ methods. Since these methods are generic and applicable to any GPCR, they should be useful in predicting the structures of other GPCRs and the binding site of ligands to these proteins.

Mutagenesis at the human tachykinin NK 2 receptor to define the binding site of a novel class of antagonists

The pharmacological profile of novel antagonists endowed with high affinity for the human tachykinin NK 2 receptor is presented. MEN13918 (Nγ{Nα[Nα(benzo[ b]thiophen-2-yl)carbonyl]-1-aminocyclohexan-1-carboxy]- d-phenylalanyl}-3- cis-aminocyclohexan-1-carboxylic-acid- N-(1 S,2 R)-2-aminocyclohexyl)amide trifluoroacetate salt) and MEN14268 (Nα[Nα(benzo[ b]thiophen-2-yl)carbonyl)-1-aminocyclopentane-1-carboxyl]- d-phenylalanine- N-[3(morpholin-4-yl)propyl]amide trifluoroacetate salt) were more potent in blocking neurokinin A (NKA, His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH 2) induced contraction in human, which induced greater contraction in human (p K B 9.1 and 8.3) than rat (p K B 6.8 and <6) urinary bladder smooth muscle preparation in vitro. In agreement with functional data, in membrane preparations of CHO cells stably expressing the human NK 2 receptors, both MEN13918 and MEN14268 potently inhibited the binding of agonist ([ 125I]NKA, K i 0.2 and 2.8 nM) and antagonist ([ 3H]nepadutant, K i 0.1 and 2.2 nM, [ 3H]SR48968 K i 0.4 and 6.9 nM) radioligands. Using site-directed mutagenesis and radioligands binding we identified six residues in the transmembrane (TM) helices that are critical determinants for the studied antagonists affinity. To visualize these experimental findings, we constructed a homology model based on the X-ray crystal structure of bovine rhodopsin and suggested a possible binding mode of these newly discovered antagonist ligands to the human tackykinin NK 2 receptor. Both MEN13918 and MEN14268 bind amongst TM4 (Cys167Gly), TM5 (Tyr206Ala), TM6 (Tyr266Ala, Phe270Ala), and TM7 (Tyr289Phe, Tyr289Thr). MEN13918 and MEN14268 diverging binding profile at Y289 mutations in TM7 (Tyr289Phe, Tyr289Thr) suggests a relation of their different chemical moieties with this residue. Moreover, the different influence on binding of these two ligands by mutations located deep along the inner side of TM6 (Phe270Ala, Tyr266Ala, Trp263Ala) indicates a nonequivalent positioning, although occupying the same binding crevice. Furthermore, binding data indicate the Ile202Phe mutation, which mimics the wild-type rat NK 2 receptor sequence, as a species selectivity determinant. In summary, data with mutant receptors describe, for these new tachykinin NK 2 receptor antagonists, a binding site which is partially overlapping either with that of the cyclized peptide antagonist nepadutant (cyclo-{[Asn(β- d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2β-5β)} or the nonpeptide antagonist SR48968 ((S)- N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide).

Novel approaches for modeling of the A1 adenosine receptor and its agonist binding site.

The present work describes the building of a human A(1) adenosine receptor (hA(1)AR) model, based on the X-ray crystal structure of bovine rhodopsin, and its use as a basis for the investigation of some important structural characteristics of the receptor. One of the issues investigated was the protonation position of two histidine residues known to influence ligand binding, with protonation of His251 (6.52) in epsilon position and His278 (7.43) in delta position showing the best agreement with experimental evidence. The model was also used to study the position and structural role of water molecules present in the helical bundle. Finally, the binding site location and the ligand docking were investigated using an objective strategy. A suitable site for the binding of the ribose moiety of adenosine was first postulated and further confirmed by means of a novel chemometric strategy based on GRIND descriptors. Using this position as an anchor point, the binding of adenosine was studied by docking and molecular dynamics simulations obtaining two putative binding positions in good agreement with experimental data.

Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca2+-sensing Receptor

A model of the Ca2+-sensing receptor (CaSR) seven transmembrane domains was constructed based on the crystal structure of bovine rhodopsin. This model was used for docking (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calhex 231), a novel potent negative allosteric modulator that blocks (IC50 = 0.39 microm) increases in [3H]inositol phosphates elicited by activating the human wild-type CaSR transiently expressed in HEK293 cells. In this model, Glu-8377.39 plays a pivotal role in anchoring the two nitrogen atoms of Calhex 231 and locating the aromatic moieties in two adjacent hydrophobic pockets delineated by transmembrane domains 3, 5, and 6 and transmembrane domains 1, 2, 3, and 7, respectively. To demonstrate its validity, we have mutated selected residues and analyzed the biochemical and pharmacological properties of the mutant receptors transfected in HEK293 cells. Two receptor mutations, F684A3.32 and E837A7.39, caused a loss of the ability of Calhex 231 to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three other mutations, F688A3.36, W818A6.48, and I841A7.43, produced a marked increase in the IC50 of Calhex 231 for the Ca2+ response, whereas L776A5.42 and F821A6.51 led to a decrease in the IC50. Our data validate the proposed model for the allosteric interaction of Calhex 231 with the seven transmembrane domains of the CaSR. Interestingly, the residues at the same positions have been shown to delimit the antagonist-binding cavity of many diverse G-protein-coupled receptors. This study furthermore suggests that the crystal structure of bovine rhodopsin exhibits sufficient mimicry to the ground state of a very divergent class 3 receptor to predict the interaction of antagonists with the heptahelical bundle of diverse G-protein-coupled receptors.

Molecular modelling studies on the ORL1-receptor and ORL1-agonists.

The ORLI (opioid receptor like 1)- receptor is a member of the family of rhodopsin-like G protein-coupled receptors (GPCR) and represents an interesting new therapeutical target since it is involved in a variety of biomedical important processes, such as anxiety, nociception, feeding, and memory. In order to shed light on the molecular basis of the interactions of the GPCR with its ligands, the receptor protein and a dataset of specific agonists were examined using molecular modelling methods. For that purpose, the conformational space of a very potent non-peptide ORL1-receptor agonist (Ro 64-6198) with a small number of rotatable bonds was analysed in order to derive a pharmacophoric arrangement. The conformational analyses yielded a conformation that served as template for the superposition of a set of related analogues. Structural superposition was achieved by employing the program FlexS. Using the experimental binding data and the superposition of the ligands, a 3D-QSAR analysis applying the GRID/GOLPE method was carried out. After the ligand-based modelling approach, a 3D model of the ORL1-receptor has been constructed using homology modelling methods based on the crystal structure of bovine rhodopsin. A representative structure of the model taken from molecular dynamics simulations was used for a manual docking procedure. Asp-130 and Thr-305 within the ORL1-receptor model served as important hydrophilic interaction partners. Furthermore, a hydrophobic cavity was identified stabilizing the agonists within their binding site. The manual docking results were supported using FlexX, which identified the same protein-ligand interaction points.

Evidence of the residues involved in ligand recognition in the second extracellular loop of the prostacyclin receptor characterized by high resolution 2D NMR techniques

In previous studies, we have determined the solution structure of the second extracellular loop (eLP 2) of the human thromboxane A 2 receptor (TP) and identified the residues in the eLP 2 domain involved in ligand recognition, by using a combination of approaches including a constrained synthetic peptide, 2D NMR spectroscopy, and recombinant proteins. These findings led us to hypothesize that the specific ligand recognition sites may be localized in the eLP 2 for all the prostanoid receptors. To test this hypothesis, we have investigated the ligand recognition site for another prostanoid receptor, the prostacyclin receptor (IP), which mediates an opposite biological function compared to that of the TP receptor. The identification of the interaction between the IP receptor and its agonist, iloprost, was achieved with a constrained synthetic peptide mimicking the eLP 2 region of the receptor. The IP eLP 2 segment was designed and synthesized to form a constrained loop, using a homocysteine disulfide bond connecting the ends of the peptide, based on the distance predicted from the IP receptor model created by homology modeling using the crystal structure of bovine rhodopsin as a template. The evidence of the constrained IP eLP 2 interaction with iloprost was found by the identification of the conformational changes of the eLP 2 induced by iloprost using fluorescence spectroscopy, and was further confirmed by 1D and 2D 1H NMR experiments. In addition, the IP eLP 2-induced structure of iloprost in solution was elucidated through a complete assignment of the 2D 1H NMR spectra for iloprost in the presence of the IP eLP 2 segment. In contrast, no ordered structure was observed in the 2D 1H NMR experiments for iloprost alone in solution. These studies not only identified that the eLP 2 segment of the IP receptor is involved in ligand recognition, but also solved the 3D solution structure of the bound-form of iloprost, which could be used to study the receptor–ligand interaction in structural terms.

Structural changes in lumirhodopsin and metarhodopsin I studied by their photoreactions at 77 K.

The functional process of rhodopsin is initiated by cis-trans photoisomerization of the retinal chromophore. One of the primary intermediates, bathorhodopsin (Batho), is stable at 77 K, and structural changes in Batho are limited around the chromophore. Then, relaxation of Batho leads to helix opening at the cytoplasmic surface in metarhodopsin II (Meta II), which allows activation of a G protein transducin. Two intermediates, lumirhodopsin (Lumi) and metarhodopsin I (Meta I), appear between Batho and Meta II, and can be stabilized at 200 and 240 K, respectively. A photoaffinity labeling experiment reported that formation of Lumi accompanied flip-over of the beta-ionone ring of the retinal chromophore so that the ring portion was attached to Ala169 of helix IV [Borhan, B., Souto, M. L., Imai, H., Shichida, Y., and Nakanishi, K. (2000) Science 288, 2209-2212]. According to the crystal structure of bovine rhodopsin, the distance between the labeled C3 atom of the chromophore and Ala169 was >15 A [Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745]. These facts suggest that global protein structural changes such as helix motions take place in Lumi. In the study presented here, Lumi and Meta I are illuminated at 77 K, and protein structural changes are probed by Fourier transform infrared (FTIR) spectroscopy. We found that Lumi can be photoconverted to rhodopsin at 77 K from the IR spectral analysis of the photoproducts of Lumi. In contrast, more complex spectra were obtained for the photoproducts of Meta I at 77 K, implying that the protein structure of Meta I is considerably altered so as not to be reverted to the original state at 77 K. Thus, these photoreaction experiments with Lumi and Meta I at 77 K suggested the presence of global protein structural changes in the process between them. We concluded that the helix motions do not occur at Lumi, but at Meta I, and the flip-over of the beta-ionone ring reported by the photoaffinity labeling takes place through the specific reaction channel without a change in the global structure.

Solution structure of the third extracellular loop of human thromboxane A 2 receptor

The extracellular domains of the thromboxane A 2 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-Å 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 (eLP 3, 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 β turn from residues 278 to 281. It also shows a distance of 9.45 Å 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 eLP 3 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.

Homology model of the CB1 cannabinoid receptor: sites critical for nonclassical cannabinoid agonist interaction.

Association of cannabimimetic compounds such as cannabinoids, aminoalkylindoles (AAIs), and arachidonylethanolamide (anandamide) with the brain cannabinoid (CB(1)) receptor activates G-proteins and relays signals to regulate neuronal functions. A CB(1) receptor homology model was constructed using the published x-ray crystal structure of bovine rhodopsin (Palczewski et al., Science, 2000, Vol. 289, pp. 739-745) in the conformation most likely to represent the "high-affinity" state for agonist binding to G-protein coupled receptors (GPCRs). A molecular docking approach that combined Monte Carlo and molecular dynamics simulations was used to identify the putative binding conformations of nonclassical cannabinoid agonists, including AC-bicyclic CP47497 and CP55940, and ACD-tricyclic CP55244. Placement of these ligands was based upon the assumption of a critical hydrogen bond between the A-ring OH and the side chain N of Lys192 in transmembrane helix 3. We evaluated two alternative binding conformations, C3-in and C3-out, denoting the directionality of the ligand C3 side chain within the receptor with respect to the inside or the outside of the cell. Assuming both the C3-in or C3-out conformation, the calculated ligand-receptor binding energy (DeltaE(bind)) was correlated with the experimentally observed binding affinity (K(i)) for a series of nonclassical cannabinoid agonists. The C3-in conformation was marginally better than the alternative C3-out conformation in predicting the rank order of the tested nonclassical cannabinoid analogs. Adopting the C3-in conformation due to the greater number of receptor interactions with known pharmacophoric elements of the ligand, key residues were identified comprising the presumed hydrophobic pocket that interacts with the C3 side chain of cannabinoid agonists. Key hydrogen bonds would form between both K3.28(192) and E(258) and the A-ring OH, and between Q(261) and the C-ring C-12 hydroxypropyl. In summary, the present study represents one of the first attempts to construct a homology model of the CB(1) cannabinoid receptor based upon the published bovine rhodopsin x-ray crystal structure and to elucidate the putative ligand binding site for nonclassical cannabinoid agonists. We postulated sites of the CB(1) receptor critical for the ligand interaction, including the hydrophobic pocket interacting with the key pharmacophoric moiety, the C3 side chain. More work is needed to delineate between two alternative (and possibly other) binding conformations of the nonclassical cannabinoid ligands within the CB(1) receptor. The present study provides a consistent framework for further investigation of the CB(1) receptor-ligand interaction and for the study of CB(1) receptor activation.

2- and 8-alkynyladenosines: conformational studies and docking to human adenosine A 3 receptor can explain their different biological behavior

Adenosine (Ado) derivatives substituted at the C2 position with an alkynyl chain are endowed with high affinity for A 1, A 2A and A 3 human adenosine receptors, while being less active at the low affinity A 2B subtype. On the other hand, the introduction of an alkynyl chain at the C8 position of adenosine is detrimental for the affinity and potency at A 1, A 2A, and A 2B receptors, while is more tolerated by the A 3 receptor. The evaluation of the stimulation of [ 35 S ]GTPγS binding revealed that 2-alkynyladenosines behave as adenosine receptors agonists while, on the contrary, 8-alkynyladenosines behave as antagonists. With this work we demonstrated, by means of an NMR-based and a computational conformational analysis, that 8-alkynyladenosines, differently from 2-alkynyladenosines, cannot adopt the sugar-base anti conformation required for adenosine receptor activation. Furthermore, using the recently reported X-ray crystal structure of bovine rhodopsin as template, we built a 3D model of the seven transmembrane domains of the human adenosine A 3 receptor with the homology modeling. After identification of the binding site we carried out docking experiments, demonstrating that the two class of molecules have different binding modes that explain their different degree of affinity and the shift of their activity from agonism to antagonism.

Comparison of the transmembrane helices of bovine rhodopsin in the crystal structure and the C α template based on cryo-electron microscopy maps and sequence analysis of the G protein-coupled receptors

G protein-coupled receptors (GPCR) are activated by a diverse array of extracellular signals, ranging from light to polypeptide molecules. The receptors propagate these signals intracellularly using G protein secondary messenger pathways. A common feature in the architecture of these receptors is their seven transmembrane domains. The first crystal structure of a GPCR, bovine rhodopsin, has recently been solved at 2.8 Å. We compared the seven membrane-spanning helices (TMH) from the crystal structure of bovine rhodopsin with those from the low-resolution model of bovine rhodopsin based on the cryo-electron microscopy structure of frog rhodopsin developed by Dr Joyce Baldwin. The model developed by Baldwin used a consensus sequence approach to predict the rotational position of each helix with respect to the other six helices. Superposition of the entire helix bundle of the Baldwin model with the crystal structure gave a RMS difference (RMSD) of 3.2 Å for the 198 C f atoms which suggests a high level of similarity in the arrangement of the helices. Except for TMH IV (RMSD of 4.0 Å), the position of corresponding helices within the helix bundle overlapped well. The superposition of individual helices showed that the RMSD values over 3 Å in the global superposition were largely due to one or more of the following: (i) differences in the unraveling and kinks for these helices, (ii) translation of TMH perpendicular to the membrane and (iii) rotation of helices up to 31°, except for TMH IV in which an additional contribution to the RMSD came from the aforementioned observation. As other crystal structures of GPCRs become available, a comparison with the Baldwin consensus model may reveal larger differences than those observed here.

Activating Mutations of the Lutropin Choriogonadotropin Receptor in Precocious Puberty

The human receptor for lutropin and chorionic gonadotropin (LHR) is a member of the G-protein-coupled receptor superfamily and plays a key role in normal and abnormal reproductive physiology. Naturally occurring mutations of the LHR gene that lead to hormone-independent signaling are associated with abnormal Leydig cell growth and precocious puberty in boys. These mutations affect 13 different residues in the LHR, with the majority clustered in the cytoplasmic half of transmembrane helix 6. Germline LHR mutations that produce constitutive activation of the G s pathway are found in cases of Leydig cell hyperplasia, while a unique somatic LHR mutation capable of activating both the G s and G q signaling pathways has been identified in Leydig cell tumors. The newly determined crystal structure of bovine rhodopsin provides a useful framework for interpreting the effects of these disease-associated LHR mutations in the context of conserved structural motifs in helix 3 and helix 7 that are known to be involved in receptor activation. As with rhodopsin, conformational signaling appears to depend on the rearrangement of key electrostatic, hydrogen-bond, and hydrophobic interactions that normally serve to stabilize the inactive LHR conformation.

Application of Covariation Analysis in 3D Structure Prediction of G-Protein Coupled Receptors

Genes encoding the large family of G binding protein coupled receptors (GPCRs) occupy perhaps 3 % of human genome. GPCRs represent one of the most important target class of proteins for drug. Over 30% of clinically marketed drugs are active at this family. Homology or comparative modeling based on the crystal structure of bovine rhodopsin [3] is currently the most frequently used for the GPCR-targeted drug design. However, the absence of significant sequence identity to the rhodopsin makes it more uncertain to predict other GPCRs’ structure. GPCRs are characterized by seven transmembrane α-helices. We have reported that seven transmembrane α-helical regions were predictable with high accuracy by adequate combination of some methods, HMM, SOSUI and Fourier Transform analysis. However, their topology is hardly predicted. To improve the accuracy of the predicted structure, the topologies of secondary structure are essential. Covariation analysis was shown to predict residue-residue contacts [2]. Therefore, we attempted covariation analysis to improve the topology prediction.

Activating Mutations of the Lutropin Choriogonadotropin Receptor in Precocious Puberty

The human receptor for lutropin and chorionic gonadotropin (LHR) is a member of the G-protein-coupled receptor superfamily and plays a key role in normal and abnormal reproductive physiology. Naturally occurring mutations of the LHR gene that lead to hormone-independent signaling are associated with abnormal Leydig cell growth and precocious puberty in boys. These mutations affect 13 different residues in the LHR, with the majority clustered in the cytoplasmic half of transmembrane helix 6. Germline LHR mutations that produce constitutive activation of the Gs pathway are found in cases of Leydig cell hyperplasia, while a unique somatic LHR mutation capable of activating both the Gs and Gq signaling pathways has been identified in Leydig cell tumors. The newly determined crystal structure of bovine rhodopsin provides a useful framework for interpreting the effects of these disease-associated LHR mutations in the context of conserved structural motifs in helix 3 and helix 7 that are known to be involved in receptor activation. As with rhodopsin, conformational signaling appears to depend on the rearrangement of key electrostatic, hydrogen-bond, and hydrophobic interactions that normally serve to stabilize the inactive LHR conformation.

G-Protein-Coupled Receptors for Light: The Three-Dimensional Structure of Rhodopsin

Two decades of structural studies on visual pigments culminated recently in the first crystal structure of bovine rhodopsin (see picture). In this archetypal G-protein-coupled receptor (GPCR) the sites of signal input, the retinal-binding site, and signal output, the G-protein-binding site, are separated by more than 40 Å. Using a multitude of biophysical and biochemical approaches, researchers now attempt to delineate a common mechanism by which signal transmission occurs in GPCRs. Furthermore, molecular details of the retinal-binding site yield first clues as to how color tuning is performed in visual pigments.

Photoisomerization in rhodopsin.

This article reviews the primary reaction processes in rhodopsin, a photoreceptive pigment for twilight vision. Rhodopsin has an 11-cis retinal as the chromophore, which binds covalently with a lysine residue through a protonated Schiff base linkage. Absorption of a photon by rhodopsin initiates the primary photochemical reaction in the chromophore. Picosecond time-resolved spectroscopy of 11-cis locked rhodopsin analogs revealed that the cis-trans isomerization of the chromophore is the primary reaction in rhodopsin. Then, generation of femtosecond laser pulses in the 1990s made it possible to follow the process of isomerization in real time. Formation of photorhodopsin within 200 fsec was observed by a transient absorption (pump-probe) experiment, which also revealed that the photoisomerization in rhodopsin is a vibrationally coherent process. Femtosecond fluorescence spectroscopy directly captured excited-state dynamics of rhodopsin, so that both coherent reaction process and unreacted excited state were observed. Faster photoreaction of the chromophore in rhodopsin than that in solution implies that the protein environment facilitates the efficient isomerization process. Such contributions of the protein residues have been monitored by infrared spectroscopy of rhodopsin, bathorhodopsin, and isorhodopsin (9-cis rhodopsin) at low temperatures. The crystal structure of bovine rhodopsin recently reported will lead to better understanding of the mechanism in future.