Structural Determinants of Cholesterol Recognition in Helical Integral Membrane Proteins.

Abstract Cholesterol is an integral component of mammalian membranes. It has been shown to modulate membrane fluidity and dynamics and alter integral membrane protein function. However, understanding the molecular mechanisms of how cholesterol impacts protein function is complicated by limited and conflicting structural data. Due to the nature of the crystallization and cryo-EM structure determination, it is difficult to distinguish between specific and biologically relevant interactions and a nonspecific association. The only widely recognized search algorithm for cholesterol-integral membrane protein interaction sites is sequence-based, i.e., searching for the so-called 'Cholesterol Recognition/interaction Amino acid Consensus' motif. While these motifs are present in numerous integral membrane proteins, there is inconclusive evidence to support their necessity or sufficiency for cholesterol binding. Here we leverage the increasing number of experimental cholesterol-integral membrane protein structures to systematically analyze putative interaction sites based on their spatial arrangement and evolutionary conservation. This analysis creates three-dimensional representations of general cholesterol interaction sites that form clusters across multiple integral membrane protein classes. We also classify cholesterol-integral membrane protein interaction sites as being either likely-specific or nonspecific. Information gleaned from our characterization will eventually enable a structure-based approach to predict and design cholesterol-integral membrane protein interaction sites.