Cholesterol-Dependent Conformational Exchange of the C-Terminal Domain of the Influenza A M2 Protein

Abstract

The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.

A Budding-Defective M2 Mutant Exhibits Reduced Membrane Interaction, Insensitivity to Cholesterol, and Perturbed Interdomain Coupling

“…39 In our earlier work, we used a truncated construct, M2-WT(23−60). To confirm that conformational exchange is indeed a property of the full-length protein, and not an artifact of using a truncation, we collected SR data for both full-length M2-WT as well as M2-5Ala.…”

“…39 The resulting SR signals were fitted with single- and biexponential decay functions using Origin software (OriginLab, Northampton, MA). Bimolecular collision rates with oxygen, W x (O 2 ), were calculated from spin−lattice relaxation rates (W e ) according to the relationship W x (O 2 ) = W e (O 2 ) − W e (N 2 ), where W e = 1/2T 1 .…”

“…DEER samples were prepared as lipodisq nanoparticles with a peptide/lipid ratio of 1:300, as previously described. 39,46 All DEER samples were prepared at a spin label concentration of 100−130 μM. Glycerol (30% w/w) was used as a cryoprotectant.…”

“…33 Our group has used SDSL-EPR to characterize the sensitivity of the M2 TM and AH domains to the surrounding environment. 20,21,36–39 We demonstrated that the M2 AH exists in conformational equilibrium and undergoes cholesterol-dependent conformational exchange. 39 We hy pothesized that this conformational exchange is relevant for M2 scission function at the edge of the viral budozone, 12,13 which is enriched in cholesterol.…”

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

“…Higher membrane fluidity and negatively charged lipids favor H37 protonation [31–33]. Cholesterol promotes the α-helical conformation [34], immobilizes tetramer rotational diffusion [35], and stabilizes tetramer assembly [36, 37]. Membrane thickness affects the tilt angle of the TM helix [33, 38–40], and negative-curvature lipids can alter the tetrameric assembly of the protein [41].…”