Importance of molecular dynamics equilibrium protocol on protein-lipid interaction near channel pore.

Multiscale molecular dynamics simulations using Martini coarse-grained (CG) and all-atom (AA) force fields are commonly used in membrane protein studies. In particular, reverse mapping an equilibrated CG model to an AA model offers an efficient way for preparing large membrane protein systems with complex protein shapes and lipid compositions. Here, we report that this hybrid CG-equilibrium-AA-production protocol may artificially increase lipid density and decrease hydration in ion channel pores walled with transmembrane gaps. To understand the origin of this conundrum, we conducted replicas of CG, AA, and CG reverse-mapped AA simulations of the pore domain of the mechanosensitive Piezo1 channel in a nonconducting conformation. Lipid/water density analysis and free energy calculations reveal that the lack of initial pore hydration allows excessive lipids to enter the upper pore lumen through gaps between pore helices during CG simulation. Due to the mismatch between CG and AA lipid kinetics, these pore lipids remain trapped in the subsequent AA simulations, despite unfavorable binding free energy. We tested several CG equilibrium protocols and found that a protocol restraining the whole lipid produces pore hydration consistent with AA results, thus eliminating this artifact for further studies of lipid gating and protein-lipid interactions.