Visualization of Protein-Lipid Interactions in Connexin-46/50 Intercellular Channels by Cryo-EM and MD-Simulation

Abstract

Cell-to-cell communication by gap junctions is facilitated by a unique macromolecular architecture, where intercellular channels directly couple the plasma membranes of two neighboring cells. In each membrane, a “hemi-channel” is formed by the oligomerization of six individual subunits (called connexins). 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 drastic lipid-induced stabilization to the channel architecture, resulting in a 3D reconstruction at 2.1 Å resolution – providing an unprecedented level of detail for the class of protein. The subunit packing within each hemi-channel is buttressed by a bouquet of highly-stabilized lipid acyl-chains. Remarkably, the stabilizing effects to the lipid-bilayer extends well beyond the annular layer of lipids and effectively immobilizes the extra-cellular leaflets of the two connected membranes. In addition, over 150 water molecules are resolved within the channel architecture. Time-averaged densities of the lipids and water obtained by MD simulation display the same pattern of stabilization seen in the CryoEM reconstruction. MD analysis suggest lipid headgroups remain highly dynamic relative to the lipid acyl-chains, rationalizing the absence of headgroup densities in the 2.1 Å CryoEM density map. 3D heterogeneity analysis of the CryoEM data identified 4 distinct classes of lipid headgroup configurations, which were also detected by network analysis of extracellular lipids in MD-simulations. Lipid and water binding sites identified in Cx46/50 are remarkably conserved across the connexin family, suggesting these stabilizing features are key contributors to the structure and function of gap junction intercellular communication.