Influenza A M2 Channel Oligomerization is Sensitive to its Chemical Environment

Abstract

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry, ion mobility spectrometry, and size-exclusion chromatography to characterize the oligomeric state of full-length AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by its local chemical environment. The monodisperse tetramer was only observed in select conditions when the antiviral drug, amantadine, was added. Native mass spectrometry of AM2 in lipid nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 forms a wider range of oligomeric states than previously thought possible, which provides new potential mechanisms of influenza pathology and pharmacology.

Lipids and EGCG Affect α-Synuclein Association and Disruption of Nanodiscs

“…If specific complexes like dimers or tetramers were being formed, we would expect to see higher relative intensities for peaks with a stoichiometry of 2 or 4, as has been previously observed with certain antimicrobial peptide 43,44 and membrane protein complexes in nanodiscs. 49,50 Instead, we observe sequential association from one to six α-syn's with no clear preferences. Complexes may still be forming, but without observable oligomeric specificity.…”

Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins

“…Recently, the availability of MS instruments designed to bring bigger and more complex systems into the gas phase-coupled with dedicated software development for the analysis of IMPs embedded in nanodiscs (Unidec)-has facilitated nMS studies of proteins in nanodiscs [51,52] or SMALPs [53]. Such progress will lead to more research on lipid-mediated oligomerization in a detergent-free environment [54,55].…”