Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

Venkata S Mandala; Shu-Yu Liao; Byungsu Kwon; Mei Hong

doi:10.1016/j.jmb.2017.05.015

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Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

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Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

Venkata S Mandala, Shu-Yu Liao, Byungsu Kwon and Mei Hong

Journal of molecular biology, Vol.429(14), pp.2192-2210

07/07/2017

DOI: 10.1016/j.jmb.2017.05.015

PMCID: PMC5761075

PMID: 28535993

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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 while W41 ensures proton conduction only from the N terminus to the C terminus and prevents reverse current under low internal pH. 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 internal pH. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type (WT) channel but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted toward 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 WT channel, consistent with faster reverse protonation from the C terminus. N NMR spectra allow the extraction of four H37 pK s and show that the pK s are more clustered in the mutant channel compared to WT 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 WT 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 WT 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.

Amino Acid Substitution

Histidine - metabolism

Hydrogen-Ion Concentration

Magnetic Resonance Spectroscopy

Mutant Proteins - chemistry

Mutant Proteins - genetics

Mutant Proteins - metabolism

Mutation, Missense

Protein Conformation - drug effects

Protein Multimerization - drug effects

Tryptophan - genetics

Tryptophan - metabolism

Viral Matrix Proteins - chemistry

Viral Matrix Proteins - genetics

Viral Matrix Proteins - metabolism

Details

Title: Subtitle: Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy
Creators: Venkata S Mandala - Massachusetts Institute of Technology
Shu-Yu Liao - Massachusetts Institute of Technology
Byungsu Kwon - Massachusetts Institute of Technology
Mei Hong - Massachusetts Institute of Technology
Resource Type: Journal article
Publication Details: Journal of molecular biology, Vol.429(14), pp.2192-2210
DOI: 10.1016/j.jmb.2017.05.015
PMID: 28535993
PMCID: PMC5761075
NLM abbreviation: J Mol Biol
ISSN: 0022-2836
eISSN: 1089-8638
Grant note: R01 GM088204 / NIGMS NIH HHS P41 EB002026 / NIBIB NIH HHS
Language: English
Date published: 07/07/2017
Academic Unit: Biochemistry and Molecular Biology
Record Identifier: 9985112880202771

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