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.

Cryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment

“…Furthermore, Y278, which interacts with Lipid 5 in all of our structures, was predicted to be involved in the interaction with neurosteroids which potentiate the GLIC activation 74 . Although the lipids in the lower leaflet interact with the important residues of GLIC, the locations and conformations of those lipids are highly conserved, showing no clear evidence of state-dependent regulatory roles, consistent with previous study 47 .…”

“…17h). Lipids 1-5 were also observed in a recent cryo-EM structure of GLIC 47 , with marginal differences in the locations of Lipids 1 and 5 (Supplementary Fig. 17j, k).…”

“…While GLIC exhibits less sensitivity to the lipid composition for the agonist-induced cation flux 63,73 , additional experimental evidence is necessary to comprehend the impact of state-dependent lipid-protein interactions. In the recent cryo-EM structure of detergent-solubilized GLIC, two lipids in the upper leaflet and three lipids in the lower leaflet were observed in each subunit, which were co-purified during sample preparation 47 . These lipids were observed upon re-processing the data from their earlier study, where no lipids were resolved, and the structures represented non-activating conformations 46 .…”

“…Notably, GLIC contains conserved cavities recognized by general anesthetics, local anesthetics, alcohols, and channel blockers [42][43][44] . However, all the deposited GLIC structures were obtained in pure or partial detergent environments, except one that was solved at low resolution using the meso crystallization technique [7][8][9][10][45][46][47] . Despite the structures being solved in detergent, few lipid molecules were found to interact with the transmembrane domain of the channel, which were believed to be extracted during solubilization or introduced by the addition of E. coli polar lipid extract during crystallization 10,11,42 .…”

Editorial: Computational and experimental insights in proton and ion translocating bioenergetic systems

“…To obtain detailed molecular insights into enzyme mechanisms, computational approaches are often applied in combination with high resolution structural data (see Lee et al, 2022 ; Bergh et al, 2024 ). On one hand, physics based classical molecular dynamics (MD) simulations can help in providing high spatial and temporal resolution of biological processes, chemical reactions, on the other hand, can be studied with QM (Quantum Mechanics)-based computational approaches.…”