Dynamics of M2 Proton Channel: Insights into the Motions of the Primary and Secondary Gates

Abstract

M2 proton channel is essential for Influenza A life cycle. The antiviral drug amantadine (AMT) used to block the M2 channel prior to the recent S31N mutation. Based on the abundant structural information on M2 we have characterized backbone and side-chain motions for Trp41 and Val27, in constructs of the transmembrane domain (M2TM) and the full length M2 protein (M2FL) reconstituted in lipid bilayers. M2 proton permeation has been shown to be dependent on the His37-Trp41 cluster, where Trp41 is the primary gate. Proton conductance directionality is lost upon Trp41 mutation. We have characterized the global rotation motion of M2TM using 2H ssNMR. Interestingly, M2TM and M2FL have very similar dynamics for the backbone and side-chain of Trp41, as indicated by the 15N powder spectra of the Trp41 site labeled in both constructs. The collapsed powder spectra of 15NεTrp41 in M2FL and M2TM indicate that Trp41 side chain undergoes motion on a fast time scale. The separated local field spectrum of aligned M2TM indicates that Trp41 side-chain is undergoing large amplitude motion that broadens the 15Nε peak. The mean orientation for the Trp41 is being determined for M2TM and M2FL, moreover the effect of AMT binding and pH activation is being addressed. We also derived the motional model of Val27 side-chain, considered a secondary gate for M2 and essential for AMT inhibition on the wild type protein. The side-chain of Val27 undergoes a two site jump motion about the Cα-Cβ bond, with unequal populations. The addition of AMT induced line broadening and a significant increase in T2 (T2apo = 27 +- 7 μs T2drug=43 +- 3 μs). The spectra of the same site on the mutant protein (M2TM_S31N-d8Val27) showed no changes upon addition of Amt.