Influenza A M2 protein conformation depends on choice of model membrane

Abstract

While crystal and NMR structures exist of the influenza A M2 protein, there is disagreement between models. Depending on the requirements of the technique employed, M2 has been studied in a range of membrane mimetics including detergent micelles and membrane bilayers differing in lipid composition. The use of different model membranes complicates the integration of results from published studies necessary for an overall understanding of the M2 protein. Here we show using site-directed spin-label EPR spectroscopy (SDSL-EPR) that the conformations of M2 peptides in membrane bilayers are clearly influenced by the lipid composition of the bilayers. Altering the bilayer thickness or the lateral pressure profile within the bilayer membrane changes the M2 conformation observed. The multiple M2 peptide conformations observed here, and in other published studies, optimistically may be considered conformations that are sampled by the protein at various stages during influenza infectivity. However, care should be taken that the heterogeneity observed in published structures is not simply an artifact of the choice of the model membrane.

Keywords: SDSL-EPR; hydrophobic mismatch; influenza A M2 protein; lateral pressure; model membrane; phosphatidylethanolamine; site-directed spin labeling.

FIGURE 1

M2 peptide sequences used for SDSL-EPR studies. Sequences correspond to the M2 protein from influenza strain A/Udorn/72 (H3N2). M2TM peptides contain residues 22-46 and are spin-labeled at the N-termini. The M2TMC peptides contain residues 23-60. M2TMC R45C/C50S peptides are spin labeled at a single cysteine site (underlined) and have the WT C50 site changed to a serine. M2TMC C50S peptides do not have a spin-label attached and are used for dilute-labeled spectra.

FIGURE 2

X-band EPR spectra of M2TMC spin-labeled at position 45 in DLPC/DLPG 4:1, DMPC/DMPG 4:1 and POPC/POPG 4:1. Peptide lipid molar ratio 1:200. Dilute-labeled spectra are shown in grey and fully labeled spectra are shown in black. Addition of M2TMC C50S was used for dilute-labeled spectra. Dilute-labeled samples have one or less spin label per tetramer. All spectra have been normalized to the same number of spins.

FIGURE 3

(A) Spin coupling (Ω) data from our previous SDSL-EPR study using N-terminus labeled M2TM peptides in PC bilayers and the current study (B) using M2TMC data from Figure 2. Both data sets provide support for a conformation change due to a change in bilayer thickness. For simplicity, only two of four peptides in the tetramer are shown in the hypothetical cartoon models shown in C and D. M2 peptides could adapt to the hydrophobic thickness of the membrane either by adjusting their tilt angle with respect to the membrane normal and/or by changing the ordering of the helical bundle from a looser tetramer, where helices make some contacts with each other, to a tighter tetramer, where helix-helix associations are maximized. These two mechanisms are not mutually exclusive and they may occur in concert with one another. For simplicity, the models shown here show a simple helix tilt mechanism.

FIGURE 4

X-band EPR spectra of M2TM in DLPC, DMPC, DOPC, and POPC membranes upon addition of cognate PE lipids. Peptide to lipid molar ration of 1:200. Dilute-labeled spectra are shown in black and fully labeled spectra are shown in red. Dilute-labeled samples have one or less spin label per tetramer. All spectra have been normalized to the same number of spins.

FIGURE 5

Calculation of spin coupling (Ω) for M2TM reconstituted into PC/PE bilayers. Ω is the ratio of the dilute-labeled spectral peak-to-peak amplitude at the central resonance line (M=0) to that of the fully labeled sample. At large spin-spin distances, Ω is approximately one (no spin-spin coupling) but increases as spin labels approach each other. The grey box indicates the limiting conformation discussed in the text.