Distance geometry calculation of G protein-coupled receptor 3D  structures.

    G protein-coupled receptors (GPCRs) are transmembrane proteins which transduce external signals to the activation of G proteins. All members of the rhodopsin-like GPCR family share common spatial structure of the transmembrane 7-a -bundle which represents the most evolutionarily conserved part of these receptors. We have developed, specifically for the modeling of GPCRs, a purely geometrical approach that relies on distance constraints, as in calculations of protein structures from NMR spectroscopy data, instead of energy optimization (Pogozheva et al., 1997).

   This approach is based upon the presence of numerous polar residues in the transmembrane segments of GPCRs. It is known that water-inaccessible polar groups of proteins have a strong tendency to form H-bonds. In transmembrane a -helices, backbone peptide groups are already paired, while the polar side-chains must interact with each other to form intra- or interhelical H-bonds. The candidate H-bonding pairs can be identified from the analysis of sequence alignments as polar residues which appear and disappear simultaneously in various GPCRs and by using approximate receptor models to exclude all spatially distant residues from the list of possible correlations. H-bonds thus identified can be applied as distance constraints for the packing of the transmembrane a -helices, using the distance geometry algorithm. Since the rhodopsin-like GPCRs share a common 3D structure of the transmembrane domain, the side-chain H-bonds from many different GPCRs can be combined in order to increase the number of simultaneously applied constraints and to calculate an "average" 7-a -bundle structure, which is subsequently used to restrain the positions of the transmembrane helices in the calculation of the 7-a-bundle for "specific" GPCRs.

    The computational procedure was organized as an iterative refinement with evolving constraints that begins with an initial model of the a -bundle and continues until each buried polar side-chain from each of the 410 GPCRs considered participate in at least one hydrogen bond in the final structure. 

    The GPCR models are consistent with experimental data that were not applied in the calculations and which can therefore be used as an independent control. The model of rhodopsin, for example, is in agreement with a vast sample of published biophysical data, such as the arrangement of a -helices in the low-resolution 3D EM maps, mapping of water- and lipid-accessible rhodopsin residues by chemical probes; identification of residues surrounding retinal by site-directed mutagenesis and cross-linking; the orientations of all-trans and 11-cis retinal relative to the membrane plane and the distances from the ligand to the intra- and extracellular surfaces, determined by linear dichroism and fluorescence quenching; reconstitution studies of opsin with synthetic retinal analogues; the "Ca -atom template" recently constructed by Baldwin et al. based on electron microscopy data (J. Mol. Biol., 272 :144-164, 1997); and many more.

 Calculated transmembrane a-bundle of GPCRs and receptor-like proteins

* GPCR-like 7TMH proteins. The coupling with G-proteins has not been demonstrated.
**   includes extracellular loops.
#  the numbering in m -receptor models corresponds to OPRM_RAT or  OPRM_MOUSE sequences.

  Structural organization of G protein-coupled receptors.

    Analysis of the atomic resolution structures of the 30 GPCRs and receptor-like proteins that have been calculated todate by the distance geometry algorithm leads to several insights into the structural organization of GPCRs.   These are illustrated in the following examples.

Interactions responsible for structural stability of the transmembrane a-bundle
1.Clustering of residues with similar polarity. 1.1 Formation of H-bonds between polar residues in the transmembrane domain. "Saturation of H-bond potential" of all polar residues in the transmembrane 7-a-bundle of the m-opioid receptor. The formation of "specific" interaction between pairs of H-bonded polar residues substantially contributes to the stability of the a-bundle in the lipid environment. Morphine (the ligand shown by orange) - also forms several H-bonds with polar receptor residues (Asp147, Asn230, Lys233, His297, Trp318)   that additionally stibilize the structure of the ligand-receptor complex .
1.2. Aromatic clusters in bovine rhodopsin.  Phe residues form clusters in the lipid-facing (green) and interior-facing (brown) sides of the helices.  Meanwhile the more polar Tyr, Trp and His residues are close to the lipid-water  membrane interface (purple).
1.3. "Polarity gradients" are observed in many proteins. For example, the highly polar pair Glu122-His221 (red-blue) located between transmembrane helices III-V of bovine rhodopsin (and also present in other rod opsins) is surrounded by a shell of Met, Cys (purple), Tyr, Trp and Phe (green) residues and then by aliphatic side chains (white). A similar Asp99-His115 pair, surrounded by aromatic and sulfur-containing residues is present in the hydrophobic interior of phospholipase A2 (4p2p  PDB file).
2. Cross-linking of residues. 2.1. Disulfide bonds. Spatially close cysteines tend to form disulfide bonds in proteins.  Eleven  possible disulfide bonds between spatially proximal cysteines, found in 105 different GPCRs from the analysis of the multisequence alignment,  were collectively applied in the calculation of the structure of the "average" receptor.
2.2. Metal-binding clusters (A).  Cys and His clusters that bind Zn+2 ions are evolutionarily designed for structure stabilization. A cluster of  four His (18, 74, 264, 268, blue) residues with geometry appropriate for Zn+2 binding has been observed after calculation  of the 7 a-bundle of squid  retinochrome.   This cluster may stabilize the structure of the extracellular domain of retinochrome, which unlike homologous opsins has a very short N-terminal tail.  This cluster is also located near the site of covalent attachment of  the all-trans-retinal chromophore (orange) to Lys271 and one of these His (His268) side chains can H-bond with Asp71, the probable counterion of  the retinal protonated Schiff 's base. It is possible that metal binding may also influence the spectral properties of the photopigment.
2.2. Metal-binding clusters (B).  Several polar residues inside the protein interior can coordinate metal ions.  The participation of Asp95 (helix II)  in binding of Na+ has been suggested from mutagenesis experiments on the d-opioid receptor (Kong et al., 1993). Asp95, other nearby polar residues, and water molecules can coordinate Na+ , which may affect  the structure of the receptor and consequently affect agonist binding.
3. Side chain packing. Close packing of side chains leads to coordinated replacements of residues in order to preserve the integrity of the transmembrane core. The appearance of the bulky Phe in crayfish rhdopsin in place of Thr64 in bovine rhodopsin is correlated with a decrease of the volume of  side chains closely packed with Thr64: Leu76 to Val and Ile307 to Ala.
Evolution of GPCR structures.
Relatedness of GPCRs and bacterial light-sensitive proteins. The optimal superposition of  the 7 a-bundle of bacteriorhodopsin (2brd.pdb, red) and bovine metarhodopsin II (1 boj.pdb, blue) gives an r.m.s.d. of 2.9 Å for 140 Ca -atoms and results in the overlap of all-trans-retinal   chromophores in both photopigments. Seven identical or functionally similar important residues in the retinal binding pockets occupy spatially similar positions.
Clustering of conserved residues.  A   "minicore" in the GPCR transmembrane domain includes 43 conserved residues that cluster by polarity: aromatic (green) and sulfur-containing (purple) residues form a cluster at the ligand-binding site (upper layer);  polar residues (Ser, Thr, Asn, Gln -yellow; Asp, Glu- red, His, Arg, Lys- blue; Tyr-green), form a central cluster and a cluster near the intracellular surface (lower layer); and a cluster of nonpolar residues (white) is seen near the middle of the receptor.  The residue numbers in the picture correspond to the bovine rhodopsin sequence.
Proximal residues in the "core" of proteins usually undergo coordinated changes during protein evolution. The change of  residue volume (see above) or polarity  usually requires the concomitant replacement of several surrounding residues to maintain the structural integrity of the protein "core".  These changes usually are not pairwise, but rather involve groups of residues. For example the conserved cluster of  aromatic residues (green) at the bottom of the ligand-binding pockets of different GPCRs (as in the d-opioid receptor,(A) are replaced by a group of polar residues (red, yellow) in glycoprotein hormone receptors (as in the lutropin/choriogonadotropin receptor, (B).	A       B
Coevolution of ligands and their binding pockets.   The complementarity of the binding cavities of receptors to their corresponding  ligands is evident from the gometrical fit, the formation of intermolecular H-bonds, and from the clustering of receptor and ligand groups with similar polarity (see structure of the binding pocket of bovine rhodospin, A).  There are similarities between the positions of relatively small agonist ligands (amines, all-trans retinal, JOM-13) inside the binding pockets of their receptors. Tyr1 of JOM-13, the tyramine moiety of amines, and the retinal polyene chain between the b-ionone ring and the 9-methyl group occupy very similar spatial positions and interact with residues in similar positions in the pockets (B).	A       B