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

Abstract

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, 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. In addition, ~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.

Fig. 1. Structure of connexin-46/50 in lipid nanodiscs by CryoEM.

a CryoEM 3D reconstruction of Cx46/50 (white) in an open-state conformation, with resolved lipid acyl chains (blue) and water molecules (red). Transparent silhouette displays the map at low contour to illustrate the dimensions of the lipid nanodisc densities, with intracellular (IC) and extracellular (EC) lipid leaflets indicated. b Zoom views of the CryoEM map and fitted atomic models, showing high-resolution features observed at 1.9 Å resolution. c Model of Cx46/50 (cylinder representation) with extracellular (EC) lipids and ordered water molecules displayed (spheres). d Cx46/50 monomer, 15 bound lipids, and 33 waters associated with each subunit. Domains labeled for transmembrane helices (TM1–4), extracellular loops (EC1–2), and N-terminal helix (NTH). The intracellular loop (ICL) and C-terminal domain (CTD) are not resolved, indicated by dotted lines.

Fig. 2. Ordered water molecules resolved in Cx46/50 by CryoEM.

a Cx46/50 subunit with segmented CryoEM density of waters overlaid in transparency (colored as in Fig. 1). Labels in a indicate position of the various zoom views, presented in b–f, showing water molecules bound to b pore-lining sites, c extracellular lipid interface, d buried intra-subunit sites, e buried subunit interface sites, and f buried cell-to-cell docking sites. In b–f, amino-acid sidechains forming hydrogen bonds to water are displayed (blue dashed lines) and labeled using Cx50 numbering.

Fig. 3. Cx46/50 induces a local phase separation to the extracellular lipid leaflet.

a Comparison of acyl-lipid density maps (blue) obtained by CryoEM and time-averaged all-atom MD simulation, overlaid onto the Cx46/50 ribbon structure (white). Inset shows a slice view (rotated 90°) of overlaid acyl-lipid densities by CryoEM (blue) and MD simulation (gray). The hexagonal packing pattern of acyl chains is indicated (solid and dotted lines) and TM helices interacting with lipid are labeled. Different subunits are indicated by suffix (a or b). b Zoom view of the acyl-lipid-binding pocket, with lipid-binding residues displayed (spheres) and labeled (Cx50 numbering). c MD snapshot of Cx50 in phosphatidylcholine (PC) lipid bilayers, with time-averaged lipid order parameter (S_CD) for each lipid indicated by shading (blue = 0.30 to white = 0.20). d Zoom view, showing an ensemble super-positioning of symmetry-related lipids obtained by MD simulation (displayed as all-atom representation, C = gray, H = white, O = red, N = blue, P = orange) occupying the MD-based lipid acyl-chain density map (blue).

Fig. 4. PC lipid configurational heterogeneity and dynamics resolved by CryoEM and MD.

a Segmented phosphatidylcholine (PC) density maps obtained by CryoEM 3D heterogeneity analysis and classification (PC Class 1—yellow, PC Class 2—orange, PC Class 3—blue). Insets show a zoom view displaying the overlapping features of resolved lipid configurations and segmented densities with fitted atomic models obtained from the three PC classes. CryoEM density for all other non-unique acyl-lipid chains, with unresolved head groups, have been omitted for clarity. b Zoom view showing Cx50 hydrogen bond interactions (blue dashed line) between the PC lipid head group and phospho-glycerol backbone. Interacting amino acids and stabilized water molecules are labeled. Yellow box—PC Class 1, orange box—PC Class 2, blue box—PC Class 3. c Illustration showing acyl-chain positions and configurational assignments resolved by CryoEM (represented as gray circles and numbered 1–15). d PC configurational classification and dwell times obtained by all-atom MD simulation, showing representative populations of stable (non-transitioning) and dynamic (transitioning) lipids. PC configurations were classified by acyl-chain occupancy in densities numbered as in c and colored uniquely (as indicated, bottom of d).