C-terminal juxtamembrane region of full-length M2 protein forms a membrane surface associated amphipathic helix

Abstract

The influenza A M2 protein is a 97-residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full-length protein. Here, the structure of the C-terminal juxtamembrane region (sites 50-60) is investigated in the full-length M2 protein using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50-60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C-terminal juxtamembrane region in full-length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full-length and truncated form of the protein.

Keywords: amphipathic helix; electron paramagnetic resonance; full-length M2 protein; site-directed spin labeling; viral budding.

Figure 1

A. Domain structure of full-length M2 protein indicating the 11 sites (50–60) that were spin-labeled in this study. B. CW X-band EPR spectra of full-length M2 protein for sites 50–60 (black lines) and previously published spectra for sites 50–56 in M2TMC (23–60) (red lines). C. Oxygen accessibilities for spin-labeled sites in full-length M2 protein (black bars) and previously published oxygen accessibilities for sites 50–56 in M2TMC (23–60) (red bars). Error bars on the ΔP_1/2 (O₂) bars are the 95% confidence intervals from the fits to the power saturation curves. M2TMC (23–60) SDSL-EPR data for sites 57–60 have not been published.