Nanodisc scaffold alters the structure of a pentameric ligand-gated ion channel revealing new conformations

Lipid nanodiscs are a powerful tool for structural and biochemical characterization of membrane proteins including ion channels. Many distinct nanodisc scaffolds are widely used, but how the nanodisc affects the structure of ion channels is poorly understood. Using single particle cryo-EM, we determined apo and agonist-bound structures of the pentameric ligand-gated ion channel (pLGIC), ELIC, in different nanodiscs: MSP1E3D1, SMA (styrene maleic acid), saposin, and spMSP1D1 (a circularized nanodisc). All structures used the same lipids (2:1:1 POPC:POPE:POPG) for reconstitution; in this lipid environment, ELIC exhibits robust responses to agonist. Strikingly, each nanodisc scaffold produced a distinct structure. In the extracellular domain (ECD) and the ECD-TMD (transmembrane domain) interface, the agonist-bound structures show incremental and concerted conformational differences associated with activation (least to most activated: SMA > MSP1E3D1 > saposin > spMSP1D1). The same trend is observed in the TMD where more activated stuctures show an outward expansion of the TMD helices. While agonist-bound structures in SMA, MSP1E3D1 and saposin show a pore with constrictions at 16’ and 9’ consistent with pre-active conformations, the structure in the circularized nanodisc, spMSP1D1, shows opening at 16’ and a constriction at 2’ consistent with a desensitized conformation. Interestingly, both apo and agonist-bound structures of ELIC in MSP1E3D1 show a uniquely contracted ECD with limited activation in the agonist-bound structure. Reconstitution of ELIC in different nanodisc scaffolds appears to produce different nanodisc diameters with the smallest being MSP1E3D1, followed by saposin, and spMSP1D1. Therefore, larger nanodiscs are associated with agonist-bound structures with greater activation. Overall, the results indicate that nanodisc scaffold significantly affects the conformation of a pLGIC, and that larger nanodiscs, achieved using a circularized nanodisc scaffold, may better mimic a native membrane environment.