Seeing Double: The Persistent Dimer‐of‐Dimers Structure of Drug Resistant Influenza A M2

The currently circulating S31N variant of the M2 proton channel of influenza A is resistant to antiviral drugs. Recently, there has been a growing concern regarding the impact of the lipid environment on the structural features of the S31N variant. The native symmetry of the M2 tetramer remains controversial. Here we show that S31N M2 persists in a dimer-of-dimers structure in different lipid preparations independent of the amount of solvating lipids up to at least 180 lipids per tetramer. NMR spectra clearly detect the characteristic resonances of the dimer-of-dimers of M2 (residues 18-60 or 18-62) reconstituted in lipids. NMR-based distance measurements indicate that two isoleucine residues with upfield shifted alpha carbon resonances, typical of extended conformations, are compatible with a particular side-chain rotameric state and helical backbone geometry. These chemical shifts are therefore compatible with the expected native transmembrane helical fold. Symmetry breaking at the pH sensing H37 residues, detected via peak doubling, is a stable feature of S31N M2 based on the reference strain Udorn/1972(H3N2). By contrast, the spectrum is dramatically altered for Columbia/2014/(H3N2) M2, which differs in sequence in the amphipathic helices. This highlights an allosteric coupling between the amphipathic helices and the pH sensing residues.