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.

SUMMARY Allosteric interactions provide precise spatiotemporal control over signaling proteins, but how allosteric activators and their targets co-evolve is poorly understood. Here, we trace the evolution of two allosteric activator motifs within the yeast scaffold protein Ste5 that specifically target the mating MAP kinase Fus3. One activator (Ste5-VWA) provides pathway insulation and dates to the divergence of Fus3 from its paralog, Kss1; a second activator (Ste5-FBD) that tunes mating behavior is, in contrast, not conserved in most lineages. Surprisingly, both Ste5 activator motifs could regulate MAP kinases that diverged from Fus3 prior to the emergence of Ste5, suggesting that Ste5 activators arose by exploiting latent regulatory features already present in the MAPK ancestor. The magnitude of this latent allosteric potential drifts widely among pre-Ste5 MAP kinases, providing a pool of hidden phenotypic diversity that, when revealed by new activators, could lead to functional divergence and the evolution of distinct signaling behaviors.

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...