Figure 2. Side view of the connexin four–helix bundle for different orientations of the TM2 helix. a) TM2 (red) oriented as proposed in [11]. The ribbons representing the other three TM helices are colored in yellow (TM1), green (TM3) and orange (TM4). TM2 residues involved in CMTX disease, according to the Swiss–Prot database (entry code P08034), are represented with sticks. Residues Arg32 (TM1) and Glu142 (TM3), which form a salt bridge exposed to the pore lumen are shown for reference. b) Same as (a) but with the TM2 orientation proposed in this work. c) RMSD fit between the initial and average conformation during the last 5 ns of simulation (white). White dashed lines highlight the kink introduced by Pro87 in TM2. doi:10.1371/journal.pone.0002614.g002 Since energy gain upon formation of solvent2exposed charged groups is rather small [24,25], it is expected that these interactions may break and form dynamically due to competition with the solvent. Nevertheless, bond occurrence was characterized by relatively prolonged time intervals (Table 2), indicating that salt bridges may play an important role in stabilizing the connexins fold. The first two salt–bridges are formed between Arg75 (TM2) with Glu residues at the extracellular side (Figure 3a), namely Glu41 (TM1) and Glu186 (TM4). The occurrence of these interactions accords with the complete connexon dissociation observed upon solubilization in dodecyl maltoside for the Cx26R75D and Cx26R75W mutants [26]. In apparent contro- versy, the Cx26R75A mutant seems to fold correctly and exhibits detergent resistance [26]. A possible explanation for this behavior is the presence of neighboring Lys187. During our simulations, this residue interacted with solvent/phospholipid molecules, but also established fluctuating salt bridges with Glu186 (Table 2). It can therefore be speculated that the stabilizing effect of the salt bridges between Arg75 and Glu186/Glu41 can be partially rescued by Lys187 in the presence of a small residue such as Ala that may confer local structural flexibility. Furthermore, connexons affected by the Cx26R75W mutation are sufficiently stable to form functional hemichannels in membranes although their properties differ from those of wild type Cx26 hemichannels, thus the absence of gap-junctional communication imparted by this mutation is not due to lack of connexon stability [27] but may Table 2. Selected salt bridge interactions calculated over the 12 ns of MD simulation (see also figure 3). H Donor– Acceptor Arg22(TM1)–Glu208(TM4) Arg32(TM1)–Glu146(TM3) Arg75(TM2)–Glu41(TM1) Arg75(TM2)–Glu186(TM4) Lys187(TM4)–Glu186(TM4) doi:10.1371/journal.pone.0002614.t002 Occupancy [%] 65 87 53 51 50 All-Atoms Connexon Model result from confined structural distortions due to missing electrostatic stabilization between Glu41 and Glu186. This may destabilize docking between apposed connexons, resulting in defective gap junction channels. The third salt bridge is formed between Arg22 (TM1) and Glu208 (TM4), at the cytoplasmic side (Figure 3b). This interaction, which is present in more than 50% of the simulation is consistent with R22G and R22P being missense mutations [28]. The fourth salt bridge between Arg32 (TM1) and Glu146 (TM3) occurs midway through the membrane, exposed to the pore lumen (Figure 3c). The formation of these two salt–bridges has been recently validated by double compensatory mutants (K22E, E209R and R32E, E146R) [29]. Hence these strong inter-helix electrostatic interactions may contribute to tie together helices TM1 to TM4 (Figure 3d). Hydrophobic interactions As shown in Figure 4, contacts between adjacent connexins, named A and B, are mediated by helices TM1 and TM3 in A, and by TM3 and TM4 in B. As previously observed, TM2 is the only helix that does not participate in inter2connexin contacts. In our simulations, these were essentially of hydrophobic nature and no firmly established electrostatic interactions were detected between adjacent connexins within the TM region. The average interface area between connexon subunits is 540 A˚2, with 15 % and 7 % of polar atoms in the interfaces of connexin subunits A and B, respectively. Notably, several aromatic residues are located within the interface (two in connexin A and 5 in connexin B, Table 3), which could further strengthen the quaternary complex through aromatic2aromatic stacking interactions. Covariance matrix Given the potential relevance of the structural fluctuations, we turned our attention to the study of the normalized covariance matrix (CM) along the MD trajectory. This study may reveal the occurrence of correlated motions between segments of the proteins that are not directly linked [30]. The normalized CM is symmetrical and ranges from 21 to 1 for anti correlated and fully correlated motions, respectively. To ease data interpretation, we calculated the CM over the TM domain. Furthermore, we reduced the redundancy in the representation by averaging over each connexin (Figure 5a) and over each connexin dimer (Figure 5b). This allowed us to examine, respectively, interactions within individual connexins and inter2connexin interactions between adjacent proteins. The diagonal values of the matrix in Figure 5a correspond to autocorrelation, which is obviously unitary, whereas the off2diagonal elements gauge interactions between distinct residues of the same connexin protein. The averaged CM values range from 0.45 to 1, indicating highly correlated motions. However, correlation is higher among TM1, 3 and 4, whereas the kink imparted by Pro87 to TM2 (see above) reduces correlation. The lowest degree of correlation occurs at Trp77, whose bulky side chain is completely exposed to the exterior of the connexon (Figure S2b). Correlation peaks indicate strong interactions among residues reported to be involved in CMTX, namely: Arg22, Ser26 and Phe29 (TM1) with Trp132, Thr134 (TM3) and His94 (TM2), all located at the cytoplasmic side. Other important correlation peaks signal interactions of Thr134 (TM3) with Ala207 (TM4) and Ala39 (TM1) with Phe149 (TM3). Of notice, mutations A39P, A39V and F149I are involved in CMTX [31]. Finally, Arg22 (TM1) and Glu208 (TM4) are correlated by salt bridge interactions (Figure 3b). The generally lower CM values in Figure 5b indicate weaker dimeric interactions. The largest peak is centered on Val152, at PLoS ONE | www.plosone.org 4 July 2008 | Volume 3 | Issue 7 | e2614