SDSL-EPR of Residues 60-70 of Influenza M2 Protein Reveals Structural Properties of a Region Involved in Viral Budding

The M2 protein is a 97-residue multifunctional transmembrane homotetrameric protein. It is found in the viral coat of the Influenza A virus, which is responsible for approximately 250,000 to 500,000 deaths annually worldwide.¹ Extensive research has elucidated M2\u27s role as an ion channel, which plays a critical role in the uncoating of the viron once it has entered the host cell. The C-terminal tail of M2 has also been shown to play a crucial role in generating curvature in the cell membrane to mediate viral budding.²³ Deletions in the C-terminal tail of the M2 protein lead to impaired viral infectivity.⁴ While the early portion of the C-terminal tail (residues 46-60) has been previously shown to be an amphipathic helix and has been extensively studied. High-resolution structural detail is limited in the region (residues 61-70), though it has been shown that these regions also play a role in viral budding. This study uses site-directed spin-label electron paramagnetic resonance (SDSL-EPR) to look at the region (residues 61-70) in the absence and presence of cholesterol. Continuous wave EPR line shapes demonstrate that this region is more mobile than amphipathic helix (46-60) suggesting that its movement is not restricted by the lipid bilayer. This assertion is also confirmed by power saturation EPR which demonstrate low ΔP_(1/2) values, suggesting that this region is not as oxygen accessible. Interestingly, a novel finding from this study is that this region has a regular conformation, which contrasts previous predictions that suggest that this region may be disordered. While further investigation is necessary, the experiments presented in this study provide a preliminary characterization to the later C-terminal domain (residues 61- 70) of the Influenza M2 protein

The Distal Cytoplasmic Tail Of The Influenza A M2 Protein Dynamically Extends From The Membrane

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61–70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle

Site-Directed Spin Labeling EPR Spectroscopy Of The Cytoplasmic Tail Of Influenza A M2

The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol