A Fully Atomistic Model of the Cx32 Connexon Sergio Pantano1,2,3*, Francesco Zonta2,3, Fabio Mammano2,3,4* 1 Institut Pasteur of Montevideo, Montevideo, Uruguay, 2 Venetian Institute of Molecular Medicine (VIMM), Padova, Italy, 3 Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), Rome, Italy, 4 Dipartimento di Fisica ‘‘G.Galilei’’, Universita` di Padova, Padova, Italy Abstract Connexins are plasma membrane proteins that associate in hexameric complexes to form channels named connexons. Two connexons in neighboring cells may dock to form a ‘‘gap junction’’ channel, i.e. an intercellular conduit that permits the direct exchange of solutes between the cytoplasm of adjacent cells and thus mediate cell–cell ion and metabolic signaling. The lack of high resolution data for connexon structures has hampered so far the study of the structure–function relationships that link molecular effects of disease–causing mutations with their observed phenotypes. Here we present a combination of modeling techniques and molecular dynamics (MD) to infer side chain positions starting from low resolution structures containing only Ca atoms. We validated this procedure on the structure of the KcsA potassium channel, which is solved at atomic resolution. We then produced a fully atomistic model of a homotypic Cx32 connexon starting from a published model of the Ca carbons arrangement for the connexin transmembrane helices, to which we added extracellular and cytoplasmic loops. To achieve structural relaxation within a realistic environment, we used MD simulations inserted in an explicit solvent–membrane context and we subsequently checked predictions of putative side chain positions and interactions in the Cx32 connexon against a vast body of experimental reports. Our results provide new mechanistic insights into the effects of numerous spontaneous mutations and their implication in connexin-related pathologies. This model constitutes a step forward towards a structurally detailed description of the gap junction architecture and provides a structural platform to plan new biochemical and biophysical experiments aimed at elucidating the structure of connexin channels and hemichannels. Citation: Pantano S, Zonta F, Mammano F (2008) A Fully Atomistic Model of the Cx32 Connexon. PLoS ONE 3(7): e2614. doi:10.1371/journal.pone.0002614 Editor: Adam Yuan, Temasek Life Sciences Laboratory, Singapore Received March 7, 2008; Accepted May 10, 2008; Published July 2, 2008 Copyright: 2008 Pantano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been funded by grants to F.M. from Fondazione Cariparo (Progetti di Eccellenza 2006), Telethon Italy (GGP05131) and the European commission FP6 Integrated Project EuroHear (LSHG-CT-20054-512063) under the Sixth Research Frame Program of The European Union. Computer simulations were performed at the CINECA supercomputer center thanks to computer-time grants from the Italian CNISM (Consorzio Nazionale Interuniversitario per le Scienze fisiche della Materia). Competing Interests: The authors have declared that no competing interests exist. * E-mail: spantano@pasteur.edu.uy (SP); fabio.mammano@unipd.it (FM) Introduction Intercellular gap junction (IGJ) channels are ubiquitous components of higher organisms that permit the direct exchange of ions and molecules up to a molecular mass of ,1 kDa between neighboring cells and thus play fundamental functions in intercellular communication between the vast majority of cell types (for comprehensive reviews, see [1,2]). IGJ channels are formed by the end–to–end non-covalent docking of two hexameric oligomers, named hemichannels or connexons [3], each provided by one of the two neighboring cells. Each one of the six subunits, named connexins [4], in the annular assembly of a connexon, comprises four hydrophobic transmembrane (TM) segments, designated TM1 to TM4 [5]. N– and C–terminal tails and one connecting loop are found within the connexin cytoplasmic region, whereas the remaining two extracellular loops permit hemichannel docking and formation of a full intercellular channel that excludes the extracellular environment [3]. Over 20 different connexin genes have been identified in mouse and human genomes [6] and spontaneous mutations in these genes have been linked to the pathogenesis of several diseases, including disorders of the heart, skin, ear and lens [7]. In particular mutations of GJB1, the gene which encodes connexin 32 (Cx32), have been implicated in some forms of the X–linked Charcot–Marie–Tooth (CMTX) disease, an inherited sensory and motor neuropathy [8]. Although a considerable effort has been devoted to elucidating structural determinants and to clarify structure/function relation- ships of these channels, only medium2 to low2resolution structures have been obtained so far (reviewed in [9]). Major contributions towards the structural determination of gap junctions have been provided by electron cryomicroscopy of channels formed by Cx43 [10]. Based on a more accurately resolved structure, Fleishman et al. [11] proposed a model for the arrangement of Ca carbon atoms in the TM helices which, owing to the wealth of data from patients with naturally occurring CMTX mutations, were mapped onto the amino acid sequence of Cx32. This choice is supported by the high degree of sequence homology in TM domains of different connexins, suggesting similar TM architecture [12]. However, it has been pointed out [9] that the structure proposed in ref. [11] may not represent a consensus model owing to disagreements in several experimental reports [13–16]. Indeed, corrections to the assignment of the helix orientations are expected due to the poor vertical resolution (,2 nm) of the electron density map. We have recently applied molecular modeling and simulation techniques to construct an atomic model of the TM part of a connexon, based on published Ca scaffold [11]. Despite the PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2614