Absolute binding affinities of phospholipids to Erwinia ligand-gated ion channel (ELIC) by streamlined alchemical free energy perturbations (SAFEP).

Abstract

An increasing number of cryo-EM structures of membrane proteins contain putative lipid binding sites with partially resolved lipid densities. In principle, these densities might be identified by computing the binding free energies of candidate ligands by alchemical free energy perturbation (FEP). In practice, however, these systems have several challenging features including lipid flexibility, system heterogeneity, and low binding affinity. Each of these confounds the distinction between the bound and unbound state: flexible molecules have large accessible volumes in conformation space; heterogeneous environments complicate the unbound state; and open, low-affinity sites mean that a “bound” ligand may still have considerably higher entropy than more in a more traditional, high-affinity binding pocket. Our SAFEP method seeks to address these problems by defining a single collective variable to define the bound state. This distance-from-bound configuration (DBC) metric reliably classifies ligand-protein conformations as “bound” or “unbound.” SAFEP has been successfully applied to the computation of absolute binding affinities of cholesterol to three GPCRs and relative binding affinities of phospholipids to two mutants of ELIC based on structural data. We have extended SAFEP to the calculation of absolute binding affinities of phospholipids. This has required additional sampling of each state and refinement to the collective variables used, especially in the unbound state. While this is somewhat more computationally expensive than computing relative free energies, absolute affinities make functional questions more accessible and mitigate the error propagation inherent to relative binding affinities. It is our hope that these methods will aid in the interpretation of similar systems.