Physics-Based Evolution of Cholesterol-Attracting Transmembrane Helices: Deciphering Cholesterol Attraction in Native Membrane Proteins

The existence of linear cholesterol-recognition motifs in alpha-helical transmembrane domains has long been debated. Our study introduces an innovative approach, evolutionary molecular dynamics (Evo-MD), which utilizes a genetic algorithm guided by coarse-grained molecular dynamics simulations. Through Evo-MD, we successfully determine the thermodynamic optimum for cholesterol attraction within isolated alpha-helical transmembrane domains (TMDs). Our investigation uncovers that cholesterol attraction in membrane proteins features a distinct and well-defined global thermodynamic optimum. This optimum arises from two key structural features: hydrophobic slenderness and hydrophobic mismatch. Additional support for these findings is provided by atomistic simulations and solid-state NMR experiments. Furthermore, we thoroughly analyze membrane protein databases and conduct live cell assays using analogous short hydrophobic sequences to explore the occurrence and feasibility of these features. Our results reveal surprising deviations from thermodynamic optimality in cholesterol attraction within native proteins. In particular, our analysis challenges the conventional belief that linear motifs such as the CRAC/CARC motif enhance cholesterol binding. Instead, we propose a rationalization that conserved aromatic residues, crucial components of the CRAC/CARC motif, likely promote dimerization by modulating membrane solubility in a cholesterol-dependent manner, rather than enhancing the binding of cholesterol. In summary, our study resolves the long-standing debate regarding linear cholesterol-recognition motifs and reveals the presence of sub-optimal cholesterol attraction in native alpha-helical transmembrane domains. These findings significantly contribute to our understanding of cholesterol-protein interactions and offer valuable insights into an alternative functional role played by conserved aromatic residues in membrane protein biology