Dimer Intermediate in the Assembly of Influenza A M2 Transmembrane Domain in Lipid Membranes

Abstract

Influenza A M2 is a single‐pass transmembrane protein, that assembles into tetrameric proton channels. This protein is a pharmacological target and knowing its structural and functional properties is of high importance. We studied the assembly of M2 transmembrane domain of (M2TMD) in DOPC/POPS membranes. To this end, we employed one of the modalities of Double Electron Electron Resonance technique (DEER), specifically spin counting based on DEER modulation depth. DEER signals were collected for a series of samples of a spin‐labeled M2TMD peptide and a progressive increase of peptide‐to‐lipid molar ratio (P/L) in the range from 1:18,800 to 1:160 and pH's 8 and 5.5. We found that DEER modulation depth strongly depends on P/L. A thorough analysis revealed a two‐stage equilibrium of M2TMD self‐association that includes initial formation of dimers at P/L smaller than 1:2,300, which then associate to form a dimer‐of‐dimers or M2 tetramers at higher P/L's. Strikingly, at P/L close to native for influenza membrane, i. e. below 1:2,500, M2TMD exists predominantly as a very stable dimer. This points to the essential role of the M2 dimer in the active channel assembly. The data obtained in detergent‐reconstituted M2 were similar to those in lipid; however oligomers were formed only at much higher peptide‐to‐detergent molar ratios. Hence, bilayer properties play a significant role in M2 self‐association and the stabilization of its functionally relevant forms. This cascade mechanism of M2 self‐oligomerization as revealed in this study could be relevant to other single‐spanning membrane proteins.