Cholesterol-Dependent Conformational Modulation and Binding Hotspots of P2RX7: Insights from Molecular Dynamics Simulations and Structural Analyses

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.