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

Abstract

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 A 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 A CRMS from the crystal structure for the ligand atoms. This predicted conformation has the carbonyl O only 2.82 A from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 0.62 A 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 A from the N of Lys-296. Making this Schiff base bond and minimizing leads to a final conformation only 2.92 A 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.

Prediction of the 3-D structure of rat MrgA G protein-coupled receptor and identification of its binding site

“…The MembStruk and HierDock protocols have also been applied to other GPCRs including bovine rhodopsin receptor [10], human dopamine D2 receptor [13], human β2 adrenergic receptor [14] and several olfactory receptors [15][16][17]. In each case the key interactions observed in the experiments are identified from our prediction, validating that these methods can predict the 3D structures of the proteins and the binding sites of strongly bound ligands to these sites.…”

“…This work builds upon our recent studies in which we first predicted the 3-D structures of mouse MrgC11 (mMrgC11) and MrgA1 (mMrgA1) receptors using the MembStruk computational method [9,10]. These structures were validated by predicting the binding sites and energies for several tetrapeptides, identifying key residues, and then experimentally confirming the expected changes in binding resulting from mutations of these residues.…”

“…This protocol has been used for several GPCRs [9,10,13,14,16,27,28], outer membrane protein A [29], and globular proteins [15,[30][31][32][33][34]. In this paper we used the modified HierDock protocol (MSCdock) described in Cho et al 2005 [35].…”

Predicted 3D structure for the human β2 adrenergic receptor and its binding site for agonists and antagonists

“…The only GPCR with an experimental 3D crystal structure is bovine rhodopsin (15,16), making this the best structure for validating MembStruk. Trabanino et al (6) showed that the TM regions of the MembStruk-predicted structure for rhodopsin agree with the crystal structure, to 2.85 Å CRMS (rms deviation in alpha carbon coordinates) for all of the main chain atoms. This good agreement with the crystal structure indicates that the MembStruk procedure predicts the helical regions reasonably well, without using any knowledge of the crystal structure.…”

Prediction of the 3D Structure and Dynamics of Human DP G-Protein Coupled Receptor Bound to an Agonist and an Antagonist

“…Using the multiple sequence alignment as input, the TM regions were predicted using TM2ndS procedure. 10 The hydrophobic maximum was chosen as the central residue (referred to as the centroid) for each helix that divides the area under the hydrophobicity curve equally. The centroid for each helix is positioned to be in the same xy plane (the midpoint of the lipid).…”