Exploring the effects of nanodiscs on the conformation of a pentameric ligand-gated ion channel.

Abstract

Recent advances in image acquisition and processing have unleashed a revolution in biomolecular structure determination via cryoelectron microscopy (cryoEM). Membrane proteins, such as the nicotinic acetylcholine receptor from the pentameric ligand-gated ion channel (pLGIC) superfamily, present challenges in structural studies due to their anisotropic and heterogenous lipid environment. In addition to the overall challenges in determining membrane protein structure, both lipid composition and membrane structure significantly affect the pLGIC conformational landscape, favoring some functional states over others. Nanodiscs, a patch of lipid bilayer surrounded by an amphipathic “scaffold”, are a standard reconstitution system for structure determination by single-particle cryoEM. Using an array of scaffolds, including the original apolipoprotein-derived membrane scaffolding protein, different size nanodiscs can be constructed to facilitate structural and biochemical studies in a quasi-native environment. Recent computational and experimental studies have shown, however, that many lipid properties, including membrane thickness, chain order parameters, and lipid diffusion, show position-dependent alterations upon incorporation into the nanodisc scaffold. To date, however, the effect of nanodisc scaffold and altered lipid properties upon a nanodisc-incorporated protein has not been systematically examined. CryoEM studies from our group have demonstrated that nanodisc scaffold choice affects the determined conformation of the pLGIC, ELIC. These nanodiscs differ in size which may be one factor influencing protein structure and dynamics. Here, we use molecular dynamics to examine how nanodisc size (small, 9 nm; medium, 11 nm; large, 13 nm) alters the transmembrane domain (TMD) structure and dynamics of ELIC, a bacterial pLGIC. Our simulation studies show that 1) the nanodisc construct and channel directly interact and 2) lipid properties are affected in a position-dependent manner leading to altered TMD dynamics. Specifically, dynamics in M4, thought to be responsible for lipid-sensing, are altered to the greatest extent.