Proton Association Constants of His 37 in the Influenza‑A M218–60 Dimer-of-Dimers

The membrane protein M2 from influenza-A forms a single-pass transmembrane helix that assembles in lipid membrane as homotetramers whose primary function is to act as a proton transporter for viral acidification. A single residue, histidine 37 (His 37), is known to be responsible for selectivity and...

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Bibliographic Details
Published in:	Biochemistry (Easton) 2014-09, Vol.53 (38), p.5987-5994
Main Authors:	Colvin, Michael T, Andreas, Loren B, Chou, James J, Griffin, Robert G
Format:	Article
Language:	eng
Subjects:	Amino Acid Sequence Histidine - chemistry Influenza A virus - metabolism Models, Molecular Nuclear Magnetic Resonance, Biomolecular Protein Structure, Secondary Protons Viral Matrix Proteins - chemistry
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Summary:	The membrane protein M2 from influenza-A forms a single-pass transmembrane helix that assembles in lipid membrane as homotetramers whose primary function is to act as a proton transporter for viral acidification. A single residue, histidine 37 (His 37), is known to be responsible for selectivity and plays an integral role in the protein’s function. We report pH-dependent 15N MAS NMR spectra of His 37 within the influenza-A proton conduction domain of M2, M218–60, which has been previously shown to be a fully functional construct and was recently determined to adopt a dimer-of-dimers structure in lipids. By extracting the ratio of [His]/[HisH+] as a function of pH, we obtained two doubly degenerate proton disassociation constants, 7.63 ± 0.15 and 4.52 ± 0.15, despite a possible maximum of four. We also report the 1HNε chemical shifts at pH 6.5 recorded at 60 kHz MAS in a CP-based 1H–15N spectrum. We were unable to detect resonances indicative of direct proton sharing among His 37 side chains when the tetramer is in the +2 state. In the neutral state, His 37 is exclusively in the τ tautomer, indicating that the δ nitrogen is protonated solely as a function of pH. We also found that the plot of [HisH+]/[His] as a function of pH is qualitatively similar to previously reported proton conduction rates, indicating that proton conduction rate is proportional to the level of histidine protonation within the channel. Two-dimensional 13C–13C and 13C–15N correlations suggest that at low pH multiple conformations are populated as the spectra broaden and eventually disappear as the acidity is increased. A second highly resolved state at low pH was not observed.
ISSN:	0006-2960 1520-4995