Cholesterol-Dependent Conformational Modulation and Binding Hotspots of P2RX7: Insights from Molecular Dynamics...

P2RX7 is an ATP-gated receptor that plays a critical role in various pathological processes including inflammation, neurodegeneration, and cancer. Cholesterol is a key modulator of membrane protein function, yet the residue-level determinants of its recognition at the purinergic receptor P2RX7 remain poorly defined. Here, we employed an integrative approach that combines data curation, molecular docking, all-atom MD simulations, and structural analyses of P2RX7 in POPC bilayers with varying cholesterol levels. Analysis of 200 experimentally determined protein-cholesterol complexes provided preliminary insights and guiding principles for evaluating interaction patterns relevant to cholesterol-P2RX7 recognition. In P2RX7, cholesterol enrichment reduced lateral lipid diffusion, increased the bilayer thickness and order, and stabilized the receptor architecture. Conformational dynamics analyses of external domain and cytoplasmic domain revealed a state-dependent "stabilized flexibility" effect, with restricted motions in the open state but enhanced fluctuations in the closed state, suggesting that cholesterol preserves functional mobility while modulating resting-state interactions. Contact mapping identified preferential interactions near the central vestibule and the TMD1-TMD2 interfaces. A weighted residue scoring scheme integrating contact persistence, solvent accessibility, pocket formation, and binding energetics prioritized high-affinity cholesterol-binding sites (CBS): CBS-I and CBS-II emerged as robust hotspots that reshuffled toward the closed state, corroborated by docking and MD simulations, while CBS-IV aligned with experimentally validated cholesterol-sensitive motifs. Together, these findings map the conformational landscape of P2RX7 and establish a residue-level framework for cholesterol-mediated regulation, revealing how hydrophobic microenvironments, polar stabilization, and binding hotspots govern cholesterol interactions. Our prioritization strategy identifies key cholesterol-binding residues, providing a systematic approach that can be applied to other protein-cholesterol systems. This work further uncovers mechanistic insights into lipid-protein allostery, highlighting sterol-sensitive sites with therapeutic potential in cancer, inflammation, and neurodegeneration.