Lipid nanodisc scaffold and size alters the structure of a pentameric ligand-gated ion channel

Abstract

Lipid nanodiscs have become the standard reconstitution system for structural and biochemical studies of membrane proteins, especially using single particle cryo-EM. We find that reconstitution of the pentameric ligand-gated ion channel (pLGIC), Erwinia ligand-gated ion channel (ELIC), in different nanodisc scaffolds (MSP1E3D1, SMA, saposin, spMSP1D1) produces distinct apo and agonist-bound structures. In the presence of agonist, different nanodiscs scaffolds produce concerted conformational changes associated with activation in ELIC, with larger nanodiscs showing more activated conformations. The effect of different nanodisc scaffolds on ELIC structure extends to the extracellular domain and agonist binding site. Molecular dynamic simulations of ELIC in small and large nanodiscs suggest that the impact of the nanodisc on ELIC structure is influenced by nanodisc size. Overall, the results indicate that the nanodisc profoundly affects the structure of a pLGIC, and suggest that larger circularized nanodiscs may be advantageous to approximate a lipid membrane environment.

Divergent mechanisms of steroid inhibition in the human ρ1 GABAAreceptor

“…In contrast, our past and current work with inhibitors favoring the resting-like state of ρ1 (TPMPA, PTX, E2) demonstrate these compounds require little or no rearrangement around the binding site, even on a local scale. These findings may reflect conditions related to cryo-EM sample preparation, favoring a desensitized-like structure not perfectly representative of the predominant physiological state 34 . Alternatively, these results may support a recent hypothesis based on detailed kinetic modeling of the α1β3γ2 GABA A receptor, where PS binding stabilizes a nonconducting state distinct from both open and desensitized 29 .…”

Divergent mechanisms of steroid inhibition in the human ρ1 GABAA receptor

“…In contrast, our past and current work with inhibitors favoring the resting-like state of ρ1 (TPMPA, PTX, E2) demonstrate these compounds require little or no rearrangement around the binding site, even on a local scale. These findings may reflect conditions related to cryo-EM sample preparation, favoring a desensitized-like structure not perfectly representative of the predominant physiological state 34 . Alternatively, these results may support a recent hypothesis based on detailed kinetic modeling of the α1β3γ2 GABAA receptor, where PS binding stabilizes a nonconducting state distinct from both open and desensitized 29 .…”

Influence of lipid bilayer on the structure of the muscle-type nicotinic acetylcholine receptor

“…Furthermore, the structure of this protein is unique among the fourteen muscle-type receptor nanodisc structures so far solved ( 7 , 8 , 17 – 19 ) in having all MX helices aligned to a planar surface, as if engaging in a specific way with a lipid bilayer. This raises the question of whether the limited dimensions and artificial lipid environment of the nanodisc ( 20 – 22 ) really can sustain a protein conformation in which properties of a cell membrane, such as lipid asymmetry and cholesterol-induced ordering, may be pivotal to the establishment of the precise physiological form. The ABC transporter MsbA exhibits widely different conformations depending on whether it is observed in nanodiscs or analyzed in the context of the native cell membrane ( 23 ), underlining the importance of interpreting with caution the structures of proteins extracted from the lipid domains in which they function.…”