Conformation and Membrane Topology of the N-Terminal Ectodomain of Influenza A M2 Protein

Abstract

The N-terminal ectodomain of the influenza A M2 protein is a target for universal influenza vaccine development and novel antiviral strategies. Despite the significance of this domain, it is poorly understood and most structural studies of the M2 protein have disregarded the N-terminal ectodomain in their analyses. Here, we report conformational properties and describe insights into the membrane topology of sites along the N-terminal ectodomain. Full-length M2 protein is embedded in lipid bilayer nanodiscs and studied using site-directed spin labeling electron paramagnetic resonance spectroscopy. Results are consistent with a turn in the middle of the ectodomain that changes in proximity to the membrane surface upon the addition of cholesterol or the antiviral drug rimantadine. Similarly to other domains of M2 protein, lineshape analysis suggests that the N-terminal ectodomain can adopt multiple conformations.

Keywords: EPR; M2 protein; ectodomain; influenza; spin labels; universal vaccine.

Figure 1

(A) The domain structure of the full-length 97-residue M2 protein. The location of spin-labeled sites (2, 4, 11, 14, 18, 21), indicated by red stars (*). The full sequence of M2 is included in Figure S1. (B) A model of a truncated homotetrameric M2 protein (residues 23–60) using previously published EPR data [11]. The work published in this paper includes ectodomain residues 1–22 not included in the ribbon model shown in 1B, but indicated by a green box. (C) A nitroxide spin label that is covalently bound to the sulfhydryl group of an introduced cysteine residue. (D) The dynamic light scattering trace of a representative M2-nanodisc sample. Stokes diameter: 11.4 ± 0.2 nm.

Figure 2

(A) X-band CW-EPR line shapes of spin-labeled M2 reconstituted into nanodiscs. Locations of sites within M2e are shown in Figure 1. (B) Mobility factors (ΔH⁻¹) as a function of spin label position. The mobility factors were calculated as the inverse linewidth of the central peak from the CW-EPR spectra shown in part A. Error bars represent the uncertainty in the position of the peak maxima and minima. (C) Access to oxygen measured by power saturation EPR as a function of spin label position. Error bars represent the 95% confidence intervals from the fits to the power saturation curves. P_1/2 (O₂) values are reported in Table S1.

Figure 3

Oxygen accessibility of sites in M2e in the presence of cholesterol ((A) orange bars) and the drug rimantadine ((B) purple bars). Green bars represent samples that do not include either cholesterol or the drug. Access to oxygen is measured by the power saturation EPR as a function of the spin label position. Error bars represent the 95% confidence intervals from the fits to the power saturation curves. The ΔP_1/2 (O₂) values used to create the bar graphs are reported in Table S1.

Figure 4

Comparison of mobility and membrane depths for the two extramembranous domains of M2. For clarity, the cartoon only shows two of the four subunits of the M2 homotetramer. The illustrated model contains values consistent with the N-terminal data reported here as well as previously published data (site 43 near the end of the TM domain, site 57 in the middle of the AH and sites 68 and 82 within the cytoplasmic tail) for the C-terminal domain of full-length M2 protein embedded in nanodiscs with the same sample composition as that used in this study [14]. Green = M2e. Yellow = TM. Blue = C-terminal AH. Pink = C-terminal tail.