G protein-coupled receptors (GPCRs) mediate our sense of vision, smell, taste, and pain. They are also involved in cell recognition and communication processes, and hence have emerged as a prominent superfamily for drug targets. Unfortunately, the atomic-level structure is available for only one GPCR (bovine rhodopsin), making it difficult to use structure-based methods to design drugs and mutation experiments. We have recently developed first principles methods (MembStruk and HierDock) for predicting structure of GPCRs, and for predicting the ligand binding sites and relative binding affinities. Comparing to the one case with structural data, bovine rhodopsin, we find good accuracy in both the structure of the protein and of the bound ligand. We report here the application of MembStruk and HierDock to ␤1-adrenergic receptor, endothelial differential gene 6, mouse and rat I7 olfactory receptors, and human sweet receptor. We find that the predicted structure of ␤1-adrenergic receptor leads to a binding site for epinephrine that agrees well with the mutation experiments. Similarly the predicted binding sites and affinities for endothelial differential gene 6, mouse and rat I7 olfactory receptors, and human sweet receptor are consistent with the available experimental data. These predicted structures and binding sites allow the design of mutation experiments to validate and improve the structure and function prediction methods. As these structures are validated they can be used as targets for the design of new receptor-selective antagonists or agonists for GPCRs.GPCR ͉ olfactory receptor ͉ ␤-adrenergic receptor ͉ endothelial differentiation gene ͉ taste receptor G protein-coupled receptors (GPCRs) mediate senses such as odor, taste, vision, and pain (1) in mammals. In addition, important cell recognition and communication processes often involve GPCRs. Indeed, many diseases involve malfunction of these receptors (2), making them important targets for drug development. Unfortunately, despite their importance there is insufficient structural information on GPCRs for structure-based drug design. This is because these membrane-bound proteins are difficult to crystallize, and the atomic-level structure has been solved only for bovine rhodopsin (3, 4). Consequently, it is important to develop theoretical methods to predict the structure and function of GPCRs (5, 6).Experimental data relevant to the function of GPCRs is available for ligand activation of GPCRs (7-15) and site-directed mutagenesis (16)(17)(18). This data has led to information about structural features in the ligand-binding regions of GPCRs (refs. 5 and 19, and references therein). Protein sequence analyses on GPCRs reveals a common protein topology consisting of a membrane-spanning seven-helix bundle, which likely accommodates the binding site for low-molecular-weight ligands. Structurally, GPCRs can be classified as (i) GPCRs with short N terminus (5-80 residues) and (ii) GPCRs with a long N-terminal ectodomain (Ϸ80-600 residues). The long N terminus of ...

Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using firstprinciples theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.W ith the implication of G protein-coupled receptor (GPCR) in many diseases (1, 2), the need to solve the highresolution 3D structure of this class of integral membrane proteins to enable structure-based drug design is an important problem in structural biology. Despite the importance of solving the structure of the GPCRs, the only experimental 3D structure available for a GPCR is bovine rhodopsin. This lack of structures is because the GPCRs are bound to the membrane, making it difficult to express in sufficient quantities for crystallization.To provide structural and ligand binding information on GPCRs, we have been developing first-principles computational techniques for predicting the 3D structure of GPCRs using only the amino acid sequence (MembStruk) and for predicting binding site and binding energy of various ligands to GPCRs (HierDock). Using these techniques, we have reported the structure of olfactory receptors (3, 4), bovine rhodopsin (4, 5), and other GPCRs (4). Dopamine neurotransmitter plays a critical role in cellular signaling processes responsible for information transfer in neurons functioning in the nervous system (6, 7). Dopamine receptors (DR) belong to the superfamily of GPCRs, and to date there are five reported sequences for the human DR with multiple isoforms for each. The DRs may be subdivided based on their pharmacological behavior into the D1-like and the D2-like subfamilies, and these are ideal targets for treating schizophrenia and Parkinson's disease; th...

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the beta(2) adrenergic receptor (beta(2)AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the beta(2)AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in beta(2)AR, namely, the breaking of the ionic lock between R131(3.50) at the intracellular end of TM3 (part of the DRY motif) and E268(6.30) on TM6, and the rotamer toggle switch on W286(6.48) on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.

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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.