Abstract
The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21−49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21−49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21−49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21−49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21−49 through increased motional ordering. In contrast to wild-type M2TMD21−49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain drug-resistant influenza strains. Thus, the inhibited M2TMD21−49 channel is a stable tetramer with a closed C-terminal exit pore. This work is aimed at contributing to the development of structure-based anti-influenza pharmaceuticals.
Highlights
Influenza pandemics cause serious health concerns for humans and for depletion of livestock
We studied the effect of drug-binding on the structure of M2TMD21−49 in DOPC/POPS by recording the double electron-electron resonance (DEER) timedomain signals for a series of spin-labeled samples, which were either amantadine-free or contained 2 mM amantadine
We aimed to characterize in greater detail the conformational transition from active to inhibited M2 transmembrane domains (M2TMD) channel and the oligomeric profile under low pH conditions, which is close to those found in endosomes, where the M2 proton channel is conductive
Summary
Influenza pandemics cause serious health concerns for humans and for depletion of livestock. In influenza A virus one such essential protein is M2, which is a small 97 amino acid integral membrane protein containing a single transmembrane helix (Figure 1A). The tetramer exhibits proton channel activity (Lamb et al, 1985; Sugrue and Hay, 1991). M2 activates at the low pH (5 to 6) of the host cell endosome, resulting in a proton influx that acidifies the viral interior leading to uncoating of the virion and release of the viral RNAs. In the process of replication, the M2 channel function is to support the transport of newly synthesized viral proteins through the Golgi apparatus by equilibrating the low intra-Golgi pH with that of the cytoplasm. The functional proton channel unit is a tetramer of M2 transmembrane domains (M2TMD) containing residues 22–46 (Ma et al, 2009). Two residues H37 and W41 are known to provide a unidirectional proton current; H37 shuttles the proton through the channel by altering its protonation state, whereas W41 serves as the channel gate (Okada et al, 2001; Wang et al, 2011)
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