Conformation of influenza AM2 membrane protein in nanodiscs and liposomes

Abstract

The influenza A M2 protein (AM2) is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle including viral assembly and budding. An atomic-level conformational understanding of this key player in the influenza life cycle could inform new antiviral strategies. For conformational studies of complex systems like the AM2 membrane protein, a multipronged approach using different biophysical methods and different model membranes is a powerful way to incorporate complementary data and achieve a fuller, more robust understanding of the system. However, one must be aware of how the sample composition required for a particular method impacts the data collected and how conclusions are drawn. In that spirit, we systematically compared the properties of AM2 in two different model membranes: nanodiscs and liposomes. Electron paramagnetic spectroscopy of spin-labeled AM2 showed that the conformation and dynamics were strikingly similar in both AM2-nanodiscs and AM2-liposomes consistent with similar conformations in both model membranes. Analysis of spin labeled lipids embedded in both model membranes revealed that the bilayer in AM2-liposomes was more fluid and permeable to oxygen than AM2-nanodiscs with the same lipid composition. Once the difference in the partitioning of the paramagnetic oxygen relaxation agent was taken into account, the membrane topology of AM2 appeared to be the same in both liposomes and nanodiscs. Finally, functionally relevant AM2 conformational shifts previously seen in liposomes due to the addition of cholesterol were also observed in nanodiscs.

Keywords: Electron paramagnetic resonance; Influenza A M2 protein; Liposomes; Membrane proteins; Nanodiscs; Oxygen concentration in membranes; Site-directed spin labeling.

Figure 1:

A. The domain structure of the full-length 97-residue AM2 protein. Location of spin-label sites (43, 57, 68 and 82) indicated by stars. B. Ensemble of models of the homotetrameric AM2 protein (23-60) built using previously published EPR data [14]. C. A cartoon model consistent with previously published experimental data [10]. For clarity only two of the four subunits of the AM2 homotetramer are shown. Location of spin-label sites (43, 57, 68 and 82) indicated by stars. D. Nitroxide spin label that is covalently bound to the sulfhydryl group of introduced cysteine residues.

Figure 2.

X-band CW-EPR line shapes of site-specific, spin-labeled AM2 reconstituted into POPC:POPG 4:1 nanodiscs (A) and liposomes (B). Locations of sites within the domain structure of AM2 are shown in Fig. 1. Cartoons of nanodiscs and liposomes are adapted from [34] and not shown to scale: Lipids (green), MSP1D1 (orange) and membrane protein (blue). Direct overlays of spectra from corresponding sites in nanodiscs and liposomes are shown in Fig. S3.

Figure 3.

Mobility and Membrane Topology of AM2 in nanodiscs and in liposomes. A. The relative mobility (inverse central line width, ΔH₀⁻¹) as a function of spin label position for nanodiscs and liposomes. The mobility factors were calculated from CW-EPR spectra shown in Fig. 2. Error bars represent the uncertainty in the position of the peak maxima and minima. B. Accessibility to oxygen measured by power saturation EPR as a function of spin label position for nanodiscs and liposomes. Error bars represent the 95% confidence intervals from the fits to the power saturation curves. ΔP_1/2(O₂) values reported in Table S1. Nanodiscs = orange bars, liposomes = green bars.

Figure 4.

A. Molecular structures of spin-labeled lipids: 16:0-5 Doxyl PC, 16:0-10 Doxyl PC B. Accessibility to oxygen measured by power saturation EPR as a function of spin label position on lipids in nanodiscs and liposomes. Error bars represent the 95% confidence intervals from the fits to the power saturation curves. ΔP_1/2(O₂) values reported in Table S1. Nanodiscs = orange bars. Liposomes = green bars.

Figure 5.

CW-EPR line shapes of site-specific, spin-labeled AM2 reconstituted into nanodiscs and liposomes both with (orange line) and without (black line) cholesterol. Locations of sites 43 and 57 within the domain structure of AM2 are shown in Fig. 1. Light gray shading highlights mobile (m) peak components. Darker gray shading highlights immobile (i) peak components. Using EPR saturation-recovery and line-shape analysis the multicomponent spectra in liposomes were previously shown to be consistent with an equilibrium involving two different conformational substates of the protein [11].