Abstract
The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homotetrameric transmembrane proton channel that acidifies the virion after endocytosis. Although influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating. Importantly, BM2 contains a second titratable histidine, His-27, in the tetrameric transmembrane domain that forms a reverse WXXXH motif with the gating tryptophan. To understand how His-27 affects the proton conduction property of BM2, we have used solid-state NMR to characterize the pH-dependent structure and dynamics of His-27. In cholesterol-containing lipid membranes mimicking the virus envelope, 15N NMR spectra show that the His-27 tetrad protonates with higher pKa values than His-19, indicating that the solvent-accessible His-27 facilitates proton conduction of the channel by increasing the proton dissociation rates of His-19. AM2 is inhibited by the amantadine class of antiviral drugs, whereas BM2 has no known inhibitors. We measured the N-terminal interhelical separation of the BM2 channel using fluorinated Phe-5. The interhelical 19F-19F distances show a bimodal distribution of a short distance of 7 Å and a long distance of 15-20 Å, indicating that the phenylene rings do not block small-molecule entry into the channel pore. These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels.
Highlights
The influenza A and B viruses are the primary cause of seasonal flu epidemics
Influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating
Critical to the viral lifecycle is the M2 protein, a single-pass, membrane spanning, homo-tetrameric proton channel responsible for acidifying the virion after endocytosis; acidification of the viral interior initiates uncoating [2]. In both influenza A and B M2 proteins, an HXXXW motif is conserved in the TM domain, in which the His is responsible for proton selectivity and acid activation [3, 4], whereas the Trp ensures asymmetric proton conduction from the N terminus to the C terminus [5, 6]
Summary
The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homotetrameric transmembrane proton channel that acidifies the virion after endocytosis. Influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating. Critical to the viral lifecycle is the M2 protein, a single-pass, membrane spanning, homo-tetrameric proton channel responsible for acidifying the virion after endocytosis; acidification of the viral interior initiates uncoating [2] In both influenza A and B M2 proteins, an HXXXW motif is conserved in the TM domain, in which the His is responsible for proton selectivity and acid activation [3, 4], whereas the Trp ensures asymmetric proton conduction from the N terminus to the C terminus [5, 6]. The Ser-9, Ile-14, Leu-18, and Ala-22 chemical shifts are insensitive to pH, whereas His-27 shows pH-dependent changes of C␣ and C chemical shifts
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