Our research focuses on the Influenza-A M2 protein, a small homotetrameric, voltage-gated ion channel. Each monomer is 97 amino acids in length, and contains a single transmembrane (TM) domain of 19 residues. This channel is very effective in transporting protons and screens out other types of ions. Although no high resolution structural data for M2 are available to date, recent NMR and CD studies of this protein in phospholipid bilayers strongly suggest that the TM region is alpha-helical and has the quaternary structure of a 4-helix bundle. The mechanisms of channel gating by M2 is also currently unknown. There are two main hypotheses regarding gating of protons. One considers the formation of a water wire through the channel and the ability of such a structure to transfer protons through the channel. The second hypothesis is based on a proton shuttle mechanism mediated by four intraluminal histidine residues forming the gate.
Our aim i n this study has been to elucidate the gating mechanism of M2, and to demonstrate the stability of a structural model of M2 in an explicit water-phospholipid bilayer system. We have performed several molecular dynamics simulations, each consisting of a trajectory at least one nanosecond long. Each simulation corresponded to a different protonation state of the histidine residues in the gate. The unprotonated and single protonated forms involved in the proton shuttle mechanism were found to be stable over the full length of the trajectory. Furthermore, the orientation of water molecules inside the channel was conducive to effective proton transfer. In contrast, the form in which all four histidine residues are protonated, required in the water-wire mechanism, was unstable and disassociated on a timescale of 400 - 700 ps. Our results demonstrate that proton shuttle involving histidine residues of the protein is the most likely mechanism of proton transport in the M2 channel.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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University of California San Francisco
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Kozak, John J; Gray, Harry B; Garza-López, Roberto A (2018) Relaxation of structural constraints during Amicyanin unfolding. J Inorg Biochem 179:135-145
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