Discovery of lipid binding sites in the ligand-gated ion channel GLIC through simulations and Cryo-EM.

Abstract

Ligand-gated ion channels play an important role in electrochemical signal transduction in neurons and other excitable cells. Aside from canonical ligands, phospholipids have been shown to bind specifically and sometimes modulate several types of channels, but the structural details of such protein-lipid interactions remain unclear. Here, we present multiple lipid interaction sites in the pentameric ligand-gated ion channel, GLIC, by integrating cryo-electron microscopy and large-scale atomistic molecular dynamics simulations together with Markov state modeling. We identified 25 lipids in the closed state of GLIC - a conformation in which, to our knowledge, lipids have not been previously resolved. Both simulations and cryo-EM data predicted, for each subunit, three lipids in the inner leaflet and two in the outer leaflet. Further, we used simulations to make predictions about lipid interactions in the open state, resulting in six sites - three in each leaflet. Interactions of particular interest include one slow-exchanging interaction with a deeply buried lipid tail in the intersubunit space, where tail saturation levels played a role in the binding pose, and where the interaction could be broken due to a single mutation. An additional lipid interaction was preferential to the open state, potentially playing a role in stabilization of this state. This work provides molecular details of GLIC-lipid contacts, especially in the understudied closed state; a testable hypotheses for state-dependent binding; and a multidisciplinary strategy for modeling protein-lipid interactions.