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.
Moro
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Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, USA.
Hoffmann
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Jacobson
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D000595:Amino Acid Sequence; D001665:Binding Sites; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1
D008024:Ligands; C546382:P2RY1 protein, human; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D019204:GTP-Binding Proteins