Structure and ligand-binding site characteristics of the human P2Y₁₁ nucleotide receptor deduced from computational modelling and mutational analysis

The P2Y₁₁-R (P2Y₁₁ receptor) is a less explored drug target. We computed an hP2Y₁₁-R (human P2Y₁₁) homology model with two templates, bovine-rhodopsin (2.6 Å resolution; 1 Å=0.1 nm) and a hP2Y₁–ATP complex model. The hP2Y₁₁-R model was refined using molecular dynamics calculations and validated by virtual screening methods, with an enrichment factor of 5. Furthermore, mutational analyses of Arg¹⁰⁶, Glu¹⁸⁶, Arg²⁶⁸, Arg³⁰⁷ and Ala³¹³ confirmed the adequacy of our hP2Y₁₁-R model and the computed ligand recognition mode. The E186A and R268A mutants reduced the potency of ATP by one and three orders of magnitude respectively. The R106A and R307A mutants were functionally inactive. We propose that residues Arg¹⁰⁶, Arg²⁶⁸, Arg³⁰⁷ and Glu¹⁸⁶ are involved in ionic interactions with the phosphate moiety of ATP. Arg³⁰⁷ is possibly also H-bonded to N⁶ of ATP via the backbone carbonyl. Activity of ATP at the F109I mutant revealed that the proposed π-stacking of Phe¹⁰⁹ with the adenine ring is a minor interaction. The mutation A313N, which is part of a hydrophobic pocket in the vicinity of the ATP C-2 position, partially explains the high activity of 2-MeS-ATP at P2Y₁-R as compared with the negligible activity at the P2Y₁₁-R. Inactivity of ATP at the Y261A mutant implies that Tyr²⁶¹ acts as a molecular switch, as in other G-protein-coupled receptors. Moreover, analysis of cAMP responses seen with the mutants showed that the efficacy of coupling of the P2Y₁₁-R with G_s is more variable than coupling with G_q. Our model also indicates that Ser²⁰⁶ forms an H-bond with P_γ (the γ-phosphate of the triphosphate chain of ATP) and Met³¹⁰ interacts with the adenine moiety.