Abstract
Lipid molecules such as cholesterol interact with the surface of integral membrane proteins (IMP) in a mode different from drug-like molecules in a protein binding pocket. These differences are due to the lipid molecule's shape, the membrane's hydrophobic environment, and the lipid's orientation in the membrane. We can use the recent increase in experimental structures in complex with cholesterol to understand protein-cholesterol interactions. We developed the RosettaCholesterol protocol consisting of (1) a prediction phase using an energy grid to sample and score native-like binding poses and (2) a specificity filter to calculate the likelihood that a cholesterol interaction site may be specific. We used a multi-pronged benchmark (self-dock, flip-dock, cross-dock, and global-dock) of protein-cholesterol complexes to validate our method. RosettaCholesterol improved sampling and scoring of native poses over the standard RosettaLigand baseline method in 91% of cases and performs better regardless of benchmark complexity. On the β2AR, our method found one likely-specific site, which is described in the literature. The RosettaCholesterol protocol quantifies cholesterol binding site specificity. Our approach provides a starting point for high-throughput modeling and prediction of cholesterol binding sites for further experimental validation.