Dolan, K. A. | Search bioRxiv

bioRxiv2022

Structure of SARS-CoV-2 M protein in lipid nanodiscs

SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other structural proteins through physical interactions and directing them to sites of viral budding. As the most abundant protein in the viral envelope and a target of patient antibodies, M is a compelling target for vaccines and therapeutics. Still, the structure of M and molecular basis for its role in virion formation are unknown. Here, we present the cryo-EM structure of SARS-CoV-2 M in lipid nanodiscs to 3.5 [A] resolution. M forms a 50 kDa homodimer that is structurally related to the SARS-CoV-2 ORF3a viroporin, suggesting a shared ancestral origin. Structural comparisons reveal how intersubunit gaps create a small, enclosed pocket in M and large open cavity in ORF3a, consistent with a structural role and ion channel activity, respectively. M displays a strikingly electropositive cytosolic surface that may be important for interactions with N, S, and viral RNA. Molecular dynamics simulations show a high degree of structural rigidity and support a role for M homodimers in scaffolding viral assembly. Together, these results provide insight into roles for M in coronavirus assembly and structure.

biophysics↗

bioRxiv2020

Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 A

Gap junctions establish direct pathways for connected cells and tissues to transfer metabolic and electrical messages1. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions2-5; however, the molecular basis for these effects remains unknown. To gain insight toward how gap junctions interact with their local membrane environment, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid membrane system, closely mimicking a native cell-to-cell junction. Structural characterization of Cx46/50 lipid-embedded channels by single particle CryoEM revealed a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 [A] resolution. Together with all-atom molecular dynamics (MD) simulations and 3D heterogeneity analysis of the ensemble CryoEM data, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet of the two opposed membranes. In addition, nearly 400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels, and demonstrates the ability of CryoEM to effectively characterize dynamical protein-lipid interactions.

biophysics↗