The V95M mutation affects similarly both Cx32 [50] and Cx26 [33] producing channels that insert in the plasma membrane but are electrically silent. V95M occurs at the interface of TM2 with the cytoplasmic loop, which is thought to be an important determinant of pH sensitivity. The previous residue, His94, also belongs to this interface and, consistently, Cx32H94Y mutants did not induce measurable conductance when paired in the homotypic configuration. This outcome has been ascribed to the high probability for at least one of the two hemichannels of being closed regardless of the transjunctional voltage [47]. Interestingly, the Cx32H94Q mutation produces functional channels that differ from wild type Cx32 only in the kinetics of the currents evoked by transjunctional voltage steps [47]. In agreement with this result, our orientation of TM2 packs both His94 and Val95 inside the helix bundle. Substitution of the bulkier Tyr at position 94 on the final averaged structure leads to important clashes. Instead no structural perturbation results from Gln replacing His at codon 94, which retains H2bonding capabilities (Figure S2h). Similarly, the longer side chain of Met at position 95 could push TM1 towards the channel lumen, obstructing the pore. In summary, although the low resolution of the electron microscopy data hinder the possibility of a definitive assignment for TM2 orientation, the one proposed here is supported by extensive mutational analysis, providing putative structure-based rationales for the effects of 16 mutants/polymorphisms along TM2 reported in nearly 30 independently published papers based on a variety of experimental techniques. More detailed structural determination or ad hoc molecular biology experiments are required to definitively solve this issue. We hope that the model presented here may constitute a structural scaffold to rationalize also the effects of other point mutations leading to pathological phenotypes. Furthermore, this theoretical framework, or possible future refinements, may aid the design of new experiments aimed at elucidating the structure/ function relationships in gap junction channels. It may additionally guide the design of novel mutants with impaired conducting or folding capabilities as a tool for selectively knocking down or knocking out connexin function. Possible extensions concern comparative modeling studies aimed at clarifying the determinants of differences among connexin isoforms. Methods Structural model of the TM section of Cx32 As mentioned above, the atomic model of the TM portion of Cx32 was constructed on the basis of a Ca model of mouse Cx32 [11] (PDB entry code: 1TXH). The Cartesian coordinates of the remaining atoms of each residue were added in their canonical conformation using the XLEAP module of AMBER 8 [61]. This procedure generated a fully atomistic model of the TM portion of Cx32 affected by a number of steric clashes and internal tensions, corresponding to a high energy state. In this configuration, the system has a marked tendency to explode driven by van der Waals repulsions among the newly introduced side chain atoms. To overcome this problem, we devised a protocol based on MD, which allows the side chains to explore the available conforma- tional space satisfying the restrictions imposed by the protein environment. Structural relaxation Starting from the initial, unstable, set of all-atoms coordinates, the system underwent 20 ps of unconstrained MD, yielding large geometrical distortions. To recover the original Ca coordinates while simultaneously finding new positions for the side chain All-Atoms Connexon Model atoms, compatible with a low energy state for the whole system, we carried out three successive rounds of constrained MD of 100 ps each, in some analogy to a simulated annealing process. Thus the TM Ca carbons were pulled back towards their original positions by harmonic constraints whose stiffness constants were set to 0.1, 1 and 10 [kcal/mol2 A˚2] in the first, second and third round, respectively. Thereafter the system underwent full energy minimization (EM), first in the presence of constraints and then unconstrained. Throughout this initial stage, EM and MD simulations were performed in the presence of implicit solvent with the Generalized Born approach [62] implemented in AMBER 8 using the Amber94 force field [63] with the following settings: cutoff distance, 1.8 nm; time–step, 2 fs; salt concentration, 0.15 M; dielectric constant, 78; temperature, 100 K using Langevin dynamics with a collision frequency of 2 ps21. The Shake algorithm was used to constraint all chemical bonds [64]. The reliability of this procedure was verified on the structure of the Potassium channel (KcsA) from Streptomyces Lividans, one of the best characterized ionic channels, solved by X-ray crystallography ([65], PDB entry code:1BL8). To this end, we used the protocol outlined above to attempt the reconstruction of the all-atoms structure starting from the sole Ca positions (see Supplementary Information). The resulting model yielded very excellent agreement, with a root mean square deviation calculated over all the heavy atoms below 2,5 A˚ and root mean square fluctuations of the side chains of 2,1 A˚ (s.d. 0,7 A˚) (see Figure S1), i.e., values comparable to standard MD simulations using well resolved structures. A comment is in order on the fact that the reliability of the above described procedure may depend strongly on the initial coordinates of the Ca atoms fitted into the low resolution electronic map. Therefore after initial relaxation of the atomistic model of the connexon we sought to reassess the positions of the TM helices within the connexin bundle. Re2evaluation of the TM2 packing within the helix bundle Since the overall reciprocal orientation of helices TM1, 3 and 4 has strong experimental support, we reassessed TM2 orientation based on a criterion that maximized surface complementarities after the initial MD relaxation. To this end we performed rigid docking of TM2 onto the all-atoms scaffold provided by helices TM1, TM3 and TM4 using the program 3D–Dock [66]. Among the several configurations sampled, we adopted the solution with the highest surface complementarity and appropriate orientation relative to the membrane plane. The results corresponds to a ,180 degrees rotation of TM2 about the helix axis relative to the configuration reported in ref. [11]. Construction of the hydrophilic loops Having achieved a stable structure for the TM domain, we undertook the construction of the remaining part of the connexon. In each connexin, the N–terminal residues from 1 to 10 were modeled as a–helices, based on NMR results derived from Cx26 fragments [67]. Residues 49 to 56 and 61 to 67, belonging to the first extra cellular loop, and residues 166 to 170 and 177 to 181, belonging to the second extra cellular loop, were modeled as double layered b2strands [68]. The C2terminal loop was truncated at Ala221 in accord with the deletion mutant used for the structural studies performed by [10]. The remaining residues for which no structural information is available were generated using the loop refinement option of the program Modeller 8.0 [69,70] while keeping the TM domain spatially constrained. It must be noticed that, although an effort has been made to stick to the fragmentary structural data available, the starting conforma- PLoS ONE | www.plosone.org 8 July 2008 | Volume 3 | Issue 7 | e2614