Insight into the Role of the Hv1 C-Terminal Domain in Dimer Stabilization.

The voltage-gated proton-selective channel (Hv1) conducts protons in response to changes in membrane potential. The Hv1 protein forms dimers in the membrane. Crystal structures of Hv1 channels have revealed that the primary contacts between the two monomers are in the C-terminal domain (CTD), which forms a coiled-coil structure. The role of Hv1-CTD in channel assembly and activity is not fully understood. Here, molecular dynamics (MD) simulations of full-length and truncated CTD models of human and mouse Hv1 channels reveal a strong contribution of the CTD to the packing of the transmembrane domains. Simulations of the CTD models highlight four fundamental interactions of the key residues contributing to dimer stability. These include salt bridges, hydrophobic interactions, hydrogen bonds, and a disulfide bond across the dimer interface. At neutral pH, salt-bridge interactions increase dimer stability and the dimer becomes less stable at acidic pH. Hydrophobic core packing of the heptad pattern is important for stability, as shown by favorable nonpolar binding free energies rather than by electrostatic components. Moreover, free-energy calculations indicate that a more uniform hydrophobic core in the coiled-coil structure of the Hv1-NIN, a channel carrying the triple mutation M234N-N235I-V236N, leads to an increase in dimer stability with respect to the wild-type. A Cys disulfide bond has a strong impact on dimer stability by holding the dimer together and facilitating the interactions described above. These results are consistent with dissociative temperatures and energy barriers of dimer dissociation obtained from the temperature-accelerated MD.