Computational Mapping of Cholesterol-Binding Landscapes in TRPV6

Abstract

Cholesterol is an important regulator of membrane protein function, yet the molecular basis of its interaction with ion channels remains incompletely understood. This is particularly relevant for TRPV6, a highly Ca2+-selective epithelial ion channel essential for systemic calcium homeostasis, whose activity is influenced by membrane lipids. Here, a multiscale computational framework combining coarse-grained molecular dynamics (CG-MD) simulations, molecular docking, and site identification by ligand competitive saturation (SILCS) was used to investigate cholesterol-interaction landscapes in TRPV6 across distinct conformational states. The three complementary approaches consistently identified recurrent cholesterol-interaction regions distributed throughout the transmembrane domain of TRPV6, particularly near the S1/S2 helices, the S4–S5 interface, and adjacent transmembrane segments. Although these interaction regions were largely conserved between open and closed conformations, cholesterol occupancy and interaction persistence differed between channel states. CG-MD simulations revealed spontaneous cholesterol-enriched regions within the membrane, SILCS identified energetically favorable interaction hotspots, and molecular docking defined preferred sterol docking sites. Together, these approaches provide a coherent description of cholesterol recognition across multiple spatial and temporal scales. Several computationally predicted interaction regions spatially overlapped with sterol-associated regions observed in cryo-EM structures of TRPV6, supporting their potential physiological relevance. Collectively, these findings indicate that cholesterol interaction with TRPV6 is distributed across multiple membrane-facing regions rather than a single dominant interaction site and is influenced by channel conformation. More broadly, this work demonstrates the value of integrating complementary computational approaches to investigate lipid–protein interactions and provides a molecular framework for understanding sterol regulation of TRPV6 and related TRP channels.