Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM

Abstract

Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside from canonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ion channels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution of these regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLIC by integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipids in the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipids were associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutant simulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, while a putative intersubunit site was preferred in open-state simulations. This work offers molecular details of GLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependent binding, and a multidisciplinary strategy for modeling protein-lipid interactions.

Constant-pH Molecular Dynamics Simulations of Closed and Open States of a Proton-gated Ion Channel

“…In terms of the simulation system setup, it was chosen to use the cryo-EM 6ZGD structure [56] rather than the X-ray 4NPQ structure [70] as a starting structure for the closed state for its slightly better resolution (4.1 Å for 6ZGD vs 4.35 Å for 4NPQ). Although an additional structure of the closed state of GLIC was recently reported to an even higher resolution (PDB ID 8ATG, 2.9 Å) [71], this model was derived from cryo-EM data collected under resting, low- and higher-activation pH conditions, rendering the interpretation of pH-sensitive changes un-certain. Regarding the choice of the membrane environment, a possible caveat is that POPC does not perfectly represent the native lipid environment.…”