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

Abstract

32Gap junctions establish direct pathways for connected cells and tissues to transfer metabolic and 33 electrical messages 1 . The local lipid environment is known to affect the structure, stability and 34 intercellular channel activity of gap junctions 2-5 ; however, the molecular basis for these effects 35 remains unknown. To gain insight toward how gap junctions interact with their local membrane 36 environment, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) 37 intercellular channels into a dual lipid membrane system, closely mimicking a native cell-to-cell 38 junction. Structural characterization of Cx46/50 lipid-embedded channels by single particle 39 CryoEM revealed a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 40 1.9 Å resolution. Together with all-atom molecular dynamics (MD) simulations and 3D 41 heterogeneity analysis of the ensemble CryoEM data, it is shown that Cx46/50 in turn imparts 42 long-range stabilization to the dynamic local lipid environment that is specific to the extracellular 43 lipid leaflet of the two opposed membranes. In addition, nearly 400 water molecules are resolved 44 in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to 45 the channel architecture. These results illustrate how the aqueous-lipid environment is integrated 46 with the architectural stability, structure and function of gap junction communication channels, 47 and demonstrates the ability of CryoEM to effectively characterize dynamical protein-lipid 48 interactions. 49 50 Main 51The connexins are a family of transmembrane proteins (21 isoforms in human) that form 52 intercellular channels for cell-to-cell communication 6 . These intercellular channels establish a 53 ~1.4 nm pore that couples the cytoplasms of neighboring cells, and enable direct passage of 54 electrical and small molecule signals (such as, ions, second messengers, hormones and 55 metabolites) 7 and therapeutic agents 8 . 10's -1000's of connexin channels may assemble 56 together to form large hexagonally packed arrays, a.k.a. plaques, known as gap junctions. In this 57 way, gap junctions enable the near instantaneous response of electrical synapses in the brain 58 and heart, and contribute to the long-range signaling and metabolic coupling of most tissues. 59Because of these fundamental roles, aberrant gap junctional coupling is associated with a variety 60 of human diseases, including blindness, deafness, skin disorders, arrhythmia, stroke and 61 cancers 9-11 . 63Gap junction intercellular communication is facilitated by a unique macromolecular architecture, 64where intercellular channels directly couple the plasma membranes of two neighboring cells. The 65 3 lipid bilayers of opposing cells are separated by a characteristic gap of ~3.5 nm 12 , a feature for 66 which these structures were first recognized in electron micrographs of cell sections 5,13 . 67Furthermore, large-scale gap junctional plaque formation is dependent upon a dense mosa...

Connexin 46 and connexin 50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N-terminal domains

“…A similar integrative approach will be key to providing mechanistic insights into the etiology of these and other cataract-associated variants and possibly other connexin-linked diseases. Lastly, we note that during the completion of this manuscript, we reported refined structural models of sheep Cx46 and Cx50 that were resolved to 1.9 Å resolution using CryoEM, in a near-native lipid environment (Flores et al, 2020). These refined models provide an unprecedented level of detail for connexin family of intercellular communication channels, and are expected to allow even more detailed investigations targeted at understanding the mechanistic principles underpinning the functional differences of these and other closely related GJ channels.…”

“…GJs with dual patch clamp on macroscopic and single channel currents. Sheep connexins were selected for this study so that we could directly align functional data with the recently described high resolution GJ structure models (Myers et al, 2018;Flores et al, 2020). From a structural point of view, the sheep orthologs of Cx46 and Cx50 show significant sequence identity in primary amino acid sequence to human (85% for Cx50 and 66% for Cx46), and over the structured domains the sequence identities are even higher (96% for Cx50 and 95% for Cx46).…”

Nanodiscs: A toolkit for membrane protein science

“…Well resolved lipids are shown in blue, pore with diameter 1.4 nm is shown as the top view on the right. Reproduced with permission from Flores et al 113 (c) Cryo‐EM structure of the Vo proton channel of yeast vacuolar ATPAse. In this structure three molecules of MSP1E3D1 scaffold stabilize the protein complex by forming rings of different diameter around middle transmembrane part and around c‐ring.…”

“…Another example of cryo‐EM resolving lipid‐protein interactions is the structure of Cx46/50, a complex of the connexins Cx46 and Cx50, each in Nanodiscs (MSP1E1 scaffold, DMPC lipids) forming a pore between two membranes, mimicking cell‐to‐cell junction with 3.5 nm gap 113 . Connexins form intercellular channels for communication of ions and small molecules between cells, often assembling into arrays, termed gap junctions, which are critical for synapse responses, and signaling.…”

Conformational changes and channel gating induced by CO2binding to Connexin26

“…3). In Cx46/50, the only other connexin structure that has been solved to date, the N-terminal helix has clear side-chain density 35,36 . In molecular dynamics simulations the N-terminal helix of Cx46-50 is more stable than its equivalent in the crystal structure of Cx26 35 .…”