All-Atoms Connexon Model Figure 3. Main electrostatic interactions from representative snapshots of the MD simulation. a) Salt bridge network between Arg75 (TM2) and Glu41 (near TM1)–Glu186 (near TM4) located at the outer leaflet of the plasma membrane. b) Interaction between Arg22 (TM1)– Glu208 (TM4) at the inner leaflet of the membrane. c) Salt bridge formed by Arg32 (TM1) and Glu146 (TM3), located nearly in the middle of the membrane and facing the pore lumen. d) The scheme highlights the putative influence of above interaction in keeping the folding of the connexin. doi:10.1371/journal.pone.0002614.g003 the extracellular end of TM3, which is a contact point with Val38 and, to a lesser extent, with Val35 in the TM1 of the adjacent connexin. Similarly, the quaternary assembly produces islands of high correlation between the C–terminal parts of TM1, TM3 and the N–terminals of TM4 (Figure 5b). The region surrounding the mid point of TM2 shows a less obvious behavior. The broad maximum is centered on Val84, which shows remarkable spatial covariance with residues belonging to other helices of contiguous protein subunits, with peaks at Phe31 (TM1), Tyr135 (TM3), Phe145 (TM3) and Val152 (TM3), despite the lack of direct contacts. This outcome accords with subtle mutations like Cx32V84I [31] and Cx26V84L [32] imparting a pathological phenotypes without major alteration in the unitary ionic conductance [33]. Discussion Reaching near2atomic resolution for the structure of biolog- ically important macromolecules, such as integral membrane proteins, is still a challenging task despite progress in specimen2- preparation techniques. In the case of connexins, breaching the resolution limits of 5210 A˚ is likely to require several years. In this interim period, molecular simulations substantiated by the wealth of molecular biology data existing in the literature may provide Figure 4. Inter connexin surface. Open-book representation of the average connexin dimer. The thick residues are those involved in protein– protein interactions reported in Table 3. Ribbons are colored by position going from red, to green and to blue from the center of the dimer. doi:10.1371/journal.pone.0002614.g004 PLoS ONE | www.plosone.org 5 July 2008 | Volume 3 | Issue 7 | e2614