Physics-based evolution of transmembrane helices reveals mechanisms of cholesterol attraction.

The existence of linear cholesterol-recognition motifs in transmembrane domains has long been debated. Evolutionary molecular dynamics (Evo-MD) simulations-genetic algorithms guided by (coarse-grained) molecular force-fields-reveal that thermodynamic optimal cholesterol attraction in isolated alpha-helical transmembrane domains occurs when multiple consecutive lysine/arginine residues flank a short hydrophobic segment. These findings are supported by atomistic simulations and solid-state NMR experiments. Our analyses illustrate that linear motifs in transmembrane domains exhibit weak binding affinity for cholesterol, characterized by sub-microsecond residence times, challenging the predictive value of linear CRAC/CARC motifs for cholesterol binding. Membrane protein database analyses suggest even weaker affinity for native linear motifs, whereas live cell assays demonstrate that optimizing cholesterol binding restricts transmembrane domains to the endoplasmic reticulum post-translationally. In summary, these findings contribute to our understanding of cholesterol-protein interactions and offer insight into the mechanisms of protein-mediated cholesterol regulation within membranes.