Table 3. Residues belonging to the TM region of the inter2connexin interface. Residue (Helix) Interface ASA % Interface ASA Connexin A ALA 19 (TM1) TRP 24 (TM1) VAL 27 (TM1) ILE 28 (TM1) PHE 31 (TM1) ARG 32 (TM1) MET 34 (TM1) VAL 35 (TM1) VAL 38 (TM1) GLU 146 (TM3) MET 150 (TM3) Connexin B TRP 133 (TM3) ILE 137 (TM3) PHE 141 (TM3) LEU 144 (TM3) PHE 145 (TM3) VAL 148 (TM3) TYR 151 (TM3) VAL 152 (TM3) PHE 190 (TM4) PHE 193 (TM4) 113.4 35.5 86.9 15.6 30.1 48.5 33.7 39.9 61.8 10.7 61.6 40.6 41.6 79.9 84.3 22.3 41.8 63.3 32.4 54.6 65.2 6.5 16 2.9 5.5 8.9 6.2 7.3 20.8 11.4 2 11.3 7.7 7.9 15.1 15.9 4.2 7.9 12 6.1 10.3 12.3 Accessible surface area (ASA) is given in A˚ 2 units. See also figure 4 doi:10.1371/journal.pone.0002614.t003 valuable tools [34] that, akin to a computational nanoscope, permit to derive images of complex and otherwise inaccessible biomolecular systems. All-Atoms Connexon Model The structural model presented here is the first attempt to provide an atomistic view for the TM domain of a connexon assembly within a realistic membrane, solvent and ionic environment. Our model, which relies on the assignment of the Ca scaffold into a low resolution map, is in agreement with a variety of experimental data derived from mutagenesis analyses, as discussed below, although small deviations from the real structure cannot be excluded at present. Comparison with the test case of the KcsA potassium channel suggest that possible errors associated to our assignments are upper bounded by the dimensions of single amino acids (see Supporting Information Text S1). Different assignments of the hydrophobic domains M1–M4 to the four helices in the cryo-EM map have been proposed [35]. Among them (reviewed in Kovacs et al., [36]), experimental data provide strong support for the positioning of TM3 and TM4 proposed in ref. [11]. That configuration is also consistent with TM1 forming a salt bridge between its Arg32 and Glu146 in TM3 (of the same connexin) [29]. However, data regarding the orientation of TM2 are of far less straightforward interpretation. Indeed, in contrast to M3 and M4, the residues in M1 and M2 are homogeneously conserved (as noted in the Figure 2a of ref. [11]) so that the orientation around their principal axis cannot be determined reliably on the basis of conservation alone. Our choice for the TM2 orientation is based on the analysis of disease2associated mutants and polymorfisms of Cx32, and the closely related Cx26 (see http://davinci.crg.es/deafness/, http:// webh01.ua.ac.be/hhh/, http://www.molgen.ua.ac.be/CMTMu- tations/ and at NCBI’s Online Mendelian Inheritance in Man server http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD = - search&DB = omim). Of notice, the TM2 of Cx32 and Cx26 differ by only two amino acids: Ser78 and Leu83 in Cx32 which are respectively replaced by Ala78 and Phe83 in Cx26. Chimerical Cx32 channels, obtained by substitution of a mutant TM2 from Cx26, show that this helix plays a critical role and is involved in voltage gating [23]. Hereafter we summarize the salient features of our analysis. Mutation of Arg75 to Trp in Cx32 (Cx32R75W), at the junction between the first extracellular loop (E1) and TM2, has twice been implicated in X–linked Charcot–Marie–Tooth disease [37,38]. The corresponding mutation, Cx26R75W, has been found in patients with dominant autosomal deafness and Figure 5. Normalized covariance matrix averaged over the six TM domains. Dark and light regions correspond to high and low correlation values respectively. White lines indicate the separation between helices. Numbers on the axes indicate the first residue of each of the TM helices, ticks are drawn every two residues. Major correlation islands commented in the text are indicated with dotted circles. To avoid redundancy, the blocks corresponding to interactions within four helices of one single connexin (a) and to interactions of helices belonging to adjacent connexins (b) are presented. doi:10.1371/journal.pone.0002614.g005 PLoS ONE | www.plosone.org 6 July 2008 | Volume 3 | Issue 7 | e2614