Assembly of influenza M2 transmembrane domain
The influenza A M2 protein is single-pass transmembrane protein that assembles in a tetramer, forming a
pH-activated proton channel (cf. Fig., upper left). It is essential for viral function. The tetramer of
M2 transmembrane domain (M2TMD), residues 22-46, is the minimal unit for proton conductance. Currently,
M2 inhibitors are the only existing anti-influenza A drugs. However, viral proliferation has been
restored by efficient natural mutagenesis that prevents drug binding and inhibition. Therefore, new
pharmaceuticals need to be developed in order to regulate M2 function and possibly suppress influenza
viral infection. The high resolution structures, provided mostly by NMR and x-ray crystallography, have
revealed several distinct conformations of M2TMD, suggesting a high structural variability. However, a
more detailed knowledge about M2-membrane interactions needs to be acquired to aid drug development.
Furthermore the mechanism of M2TMD assembly and structural alterations upon channel activation in lipid
membranes is still not fully understood. We utilized pulsed ESR spectroscopy (DEER) with protein spin-
labeling to study the M2TMD peptide (residues 21-49) in lipid membranes. A unique cysteine residue
(L46C), introduced in M2TMD, was spin-labeled with MTSL. The peptide was reconstituted in membranes of
different composition, i. e. zwitterionic or mixtures of zwitterionic and anionic lipids, as well as
variable thickness. We measured inter-spin distances and their distributions (P(r), cf. Fig., upper
right) between membrane-embedded M2TMD spin-labeled monomers in order to elucidate their assembly. Based
on the modulation depth of DEER signals, that is the signal amplitude at zero evolution time (cf. Fig.
lower left and right), and the reconstructed distance distributions, our results suggest that membrane-
associated M2TMD is polydisperse and most likely exists as monomers, dimers and tetramers. We also found
that the assembly and the structure of M2TMD oligomers are directly coupled to the membrane hydrocarbon
thickness. Moreover, anionic lipids at physiological concentration are required for M2TM channel opening
at low pH. Thus, our results contribute to the understanding of M2 properties and also to the fundamental
knowledge about folding and assembly of transmembrane helices.
Publication: Elka R. Georgieva, Haley D. Norman, Peter P. Borbat, and Jack H. Freed.
The assembly, structure and activation of influenza A M2 transmembrane domain depends on lipid membrane
thickness and composition, Biophys J, 106 (2014) pp. 249a (Abstract from the 58th
Biophysical Society Meeting).