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Demystifying P2Y1 Receptor Ligand Recognition through Docking and Molecular Dynamics Analyses.

ABSTRACT: We performed a molecular modeling analysis of 100 nucleotide-like bisphosphates and 46 non-nucleotide arylurea derivatives previously reported as P2Y1R binders using the recently solved hP2Y1R structures. We initially docked the compounds at the X-ray structures and identified the binding modes of representative compounds highlighting key patterns in the structure-activity relationship (SAR). We subsequently subjected receptor complexes with selected key agonists (2MeSADP and MRS2268) and antagonists (MRS2500 and BPTU) to membrane molecular dynamics (MD) simulations (at least 200 ns run in triplicate, simulation time 0.6-1.6 ?s per ligand system) while considering alternative protonation states of nucleotides. Comparing the temporal evolution of the ligand-protein interaction patterns with available site-directed mutagenesis (SDM) data and P2Y1R apo state simulation provided further SAR insights and suggested reasonable explanations for loss/gain of binding affinity as well as the most relevant charged species for nucleotide ligands. The MD analysis also predicted local conformational changes required for the receptor inactive state to accommodate nucleotide agonists.

SUBMITTER: Ciancetta A

PROVIDER: S-EPMC5953180 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Demystifying P2Y1 Receptor Ligand Recognition through Docking and Molecular Dynamics Analyses.

Ciancetta Antonella A O'Connor Robert D RD Paoletta Silvia S Jacobson Kenneth A KA

Journal of chemical information and modeling 20171128 12

We performed a molecular modeling analysis of 100 nucleotide-like bisphosphates and 46 non-nucleotide arylurea derivatives previously reported as P2Y1R binders using the recently solved hP2Y1R structures. We initially docked the compounds at the X-ray structures and identified the binding modes of representative compounds highlighting key patterns in the structure-activity relationship (SAR). We subsequently subjected receptor complexes with selected key agonists (2MeSADP a ...[more]

PMID: 29182323

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Project description:We conducted a mutational analysis of residues potentially involved in the adenine nucleotide binding pocket of the human P2Y1 receptor. Mutated receptors were expressed in COS-7 cells with an epitope tag that permitted confirmation of expression in the plasma membrane, and agonist-promoted inositol phosphate accumulation was assessed as a measure of receptor activity. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7 predicted by molecular modeling to be involved in ligand recognition were replaced with alanine and, in some cases, by other amino acids. The potent P2Y1 receptor agonist 2-methylthio-ATP (2-MeSATP) had no activity in cells expressing the R128A, R310A, and S314A mutant receptors, and a markedly reduced potency of 2-MeSATP was observed with the K280A and Q307A mutants. These results suggest that residues on the exofacial side of TM3 and TM7 are critical determinants of the ATP binding pocket. In contrast, there was no change in the potency or maximal effect of 2-MeSATP with the S317A mutant receptor. Alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant receptors that exhibited a 7-18-fold reduction in potency compared with that observed with the wild-type receptor. These residues thus seem to subserve a less important modulatory role in ligand binding to the P2Y1 receptor. Because changes in the potency of 2-methylthio-ADP and 2-(hexylthio)-AMP paralleled the changes in potency of 2-MeSATP at these mutant receptors, the beta- and gamma-phosphates of the adenine nucleotides seem to be less important than the alpha-phosphate in ligand/P2Y1 receptor interactions. However, T221A and T222A mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) than in diphosphate or monophosphate potency. This result may be indicative of a greater role of these TM5 residues in gamma-phosphate recognition. Taken together, the results suggest that the adenosine and alpha-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of the human P2Y1 receptor.

Project description:The urea transporter UT-B1, encoded by the SLC14A1 gene, has been hypothesized to be a significant protein whose deficiency and dysfunction contribute to the pathogenesis of bladder cancer and many other diseases. Several studies reported the association of genetic alterations in the SLC14A1 (UT-B1) gene with bladder carcinogenesis, suggesting a need for thorough characterization of the UT-B1 protein's coding and non-coding variants. This study used various computational techniques to investigate the commonly occurring germ-line missense and non-coding SNPs (ncSNPs) of the SLC14A1 gene (UT-B1) for their structural, functional, and molecular implications for disease susceptibility and dysfunctionality. SLC14A1 missense variants, primarily identified from the ENSEMBL genome browser, were screened through twelve functionality prediction tools leading to two variants D280Y (predicted detrimental by maximum tools) and D280N (high global MAF) for rs1058396. Subsequently, the ConSurf and NetSurf tools revealed the D280 residue to be in a variable site and exposed on the protein surface. According to I-Mutant2.0 and MUpro, both variants are predicted to cause a significant effect on protein stability. Analysis of molecular docking anticipated these two variants to decrease the binding affinity of UT-B1 protein for the examined ligands to a significant extent. Molecular dynamics also disclosed the possible destabilization of the UT-B1 protein due to single nucleotide polymorphism compared to wild-type protein which may result in impaired protein function. Furthermore, several non-coding SNPs were estimated to affect transcription factor binding and regulation of SLC14A1 gene expression. Additionally, two ncSNPs were found to affect miRNA-based post-transcriptional regulation by creating new seed regions for miRNA binding. This comprehensive in-silico study of SLC14A1 gene variants may serve as a springboard for future large-scale investigations examining SLC14A1 polymorphisms.

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Demystifying P2Y1 Receptor Ligand Recognition through Docking and Molecular Dynamics Analyses.

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Demystifying P2Y<sub>1</sub> Receptor Ligand Recognition through Docking and Molecular Dynamics Analyses.

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