Abstract
Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-electron microscopy (cryo-EM) densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities using experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in, e.g., cryo-EM derived membrane protein structures surrounded by an array of ancillary lipid densities.