Cheng, W. W. L. | Search bioRxiv

bioRxiv2022

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

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.

biophysics↗

bioRxiv2018

Multiple Functional Neurosteroid Binding Sites on GABAA Receptors

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While GABAA receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill-defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry, we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human 1{beta}3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the 1 and {beta}3 subunits, adjacent to the extracellular domains. An intersubunit site in the interface between the {beta}3(+) and 1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies based on these predictions indicate that both the 1 intrasubunit and {beta}3-1 intersubunit sites are critical for neurosteroid action.

neuroscience↗