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J Mol Biol. 2017 May 20. pii: S0022-2836(17)30244-9. doi: 10.1016/j.jmb.2017.05.015. [Epub ahead of print]
Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy.
Mandala VS1, Liao SY1, Kwon B1, Hong M2.
Author information
Abstract
The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH (pHout) while W41 ensures proton conduction only from the N-terminus to the C-terminus and prevents reverse current under low internal pH (pHin). Here we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low pHin. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type channel, but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted towards the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C-terminus. At low pH, the mutant channel contains more cationic tetrads than the wild-type channel, consistent with faster reverse protonation from the C-terminus. 15N NMR spectra allow the extraction of four H37 pKa's and show that the pKa's are more clustered in the mutant channel compared to wild-type M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in wild-type M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in wild-type M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants.
Copyright ? 2017. Published by Elsevier Ltd.
KEYWORDS:
Magic-angle-spinning NMR; gating; ion channels; proton dissociation equilibrium; tautomeric equilibrium
PMID: 28535993 DOI: 10.1016/j.jmb.2017.05.015
Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy.
Mandala VS1, Liao SY1, Kwon B1, Hong M2.
Author information
Abstract
The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH (pHout) while W41 ensures proton conduction only from the N-terminus to the C-terminus and prevents reverse current under low internal pH (pHin). Here we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low pHin. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type channel, but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted towards the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C-terminus. At low pH, the mutant channel contains more cationic tetrads than the wild-type channel, consistent with faster reverse protonation from the C-terminus. 15N NMR spectra allow the extraction of four H37 pKa's and show that the pKa's are more clustered in the mutant channel compared to wild-type M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in wild-type M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in wild-type M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants.
Copyright ? 2017. Published by Elsevier Ltd.
KEYWORDS:
Magic-angle-spinning NMR; gating; ion channels; proton dissociation equilibrium; tautomeric equilibrium
PMID: 28535993 DOI: 10.1016/j.jmb.2017.05.015