Identification and analysis of functionally important amino acids in human purinergic 12 receptor using a Saccharomyces cerevisiae expression system

Abstract

The purinergic 12 receptor (P2Y12) is a major drug target for anticoagulant therapies, but little is known about the regions involved in ligand binding and activation of this receptor. We generated four randomized P2Y12 libraries and investigated their ligand binding characteristics. P2Y12 was expressed in a Saccharomyces cerevisiae model system. Four libraries were generated with randomized amino acids at positions 181, 256, 265 and 280. Mutant variants were screened for functional activity in yeast using the natural P2Y12 ligand ADP. Activation results were investigated using quantitative structure-activity relationship (QSAR) models and ligand-receptor docking. We screened four positions in P2Y12 for functional activity by substitution with amino acids with diverse physiochemical properties. This analysis revealed that positions E181, R256 and R265 alter the functional activity of P2Y12 in a specific manner. QSAR models for E181 and R256 mutant libraries strongly supported the experimental data. All substitutions of amino acid K280 were completely inactive, highlighting the crucial role of this residue in P2Y12 function. Ligand-receptor docking revealed that K280 is likely to be a key element in the ligand-binding pocket of P2Y12. The results of this study demonstrate that positions 181, 256, 265 and 280 of P2Y12 are important for the functional integrity of the receptor. Moreover, K280 appears to be a crucial feature of the P2Y12 ligand-binding pocket. These results are important for rational design of novel antiplatelet agents.

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y12

“…Adenosine rings frequently interacted with the hydrophobic residues on transmembrane helix 5, namely L 184 , V 185 , and F 177 in the second extracellular loop. In agreement with previous docking studies, R 256 and K 280 were found to be critical residues in the ADP binding pocket (Deflorian and Jacobson, 2011;Ignatovica et al, 2011 259 seems to stabilize the adenine. Identification of Constitutively Active Mutants.…”

P2Y12 receptors: structure and function

“…The importance of this region of the molecule was shown also by in vitro mutagenesis studies [21,22].…”

Structure of the human P2Y12 receptor in complex with an antithrombotic drug

“…7). In both docking models, R256 6.55 , which was previously reported to be important for the activation of P2Y 12 R 24,25 , potentially makes contact with the phosphate groups of 2MeSADP. This is consistent with pharmacological and biochemical data showing that mutations of this residue affect both the number of binding sites and their affinities for radio-labelled 2MeSADP in patient platelets and in transfected CHO cells 26–28 .…”