Abstract
Influenza A and B viruses cause seasonal flu epidemics. The M2 protein of influenza B (BM2) is a membrane-embedded tetrameric proton channel that is essential for the viral lifecycle. BM2 is a functional analog of AM2 but shares only 24% sequence identity for the transmembrane (TM) domain. The structure and function of AM2, which is targeted by two antiviral drugs, have been well characterized. In comparison, much less is known about the structure of BM2 and no drug is so far available to inhibit this protein. Here we use solid-state NMR spectroscopy to investigate the conformation of BM2(1–51) in phospholipid bilayers at high pH, which corresponds to the closed state of the channel. Using 2D and 3D correlation NMR experiments, we resolved and assigned the 13C and 15N chemical shifts of 29 residues of the TM domain, which yielded backbone (φ, ψ) torsion angles. Residues 6–28 form a well-ordered α-helix, whereas residues 1–5 and 29–35 display chemical shifts that are indicative of random coil or β-sheet conformations. The length of the BM2-TM helix resembles that of AM2-TM, despite their markedly different amino acid sequences. In comparison, large 15N chemical shift differences are observed between bilayer-bound BM2 and micelle-bound BM2, indicating that the TM helix conformation and the backbone hydrogen bonding in lipid bilayers differ from the micelle-bound conformation. Moreover, HN chemical shifts of micelle-bound BM2 lack the periodic trend expected for coiled coil helices, which disagree with the presence of a coiled coil structure in micelles. These results establish the basis for determining the full three-dimensional structure of the tetrameric BM2 to elucidate its proton-conduction mechanism.
Highlights
Influenza and pneumonia cause 9 to 35 million cases of infection in humans and over 55,000 deaths each year in the US1
We chose BM2(1–51) (Fig. 1) as the construct for conformational analysis because this domain includes the TM domain required for proton conduction as well as the necessary cytoplasmic segment for membrane targeting of the protein
We were not able to cleave the N-terminal solubility tag used for protein expression; the tag is disordered and highly mobile, as it exhibits narrow signals in the 13C Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) spectrum, it does not interact with the TM helix
Summary
Influenza and pneumonia cause 9 to 35 million cases of infection in humans and over 55,000 deaths each year in the US1. Whole-cell electrophysiology data[16] showed that the two proteins have similar inward proton current at negative voltages, but at positive voltages BM2 conducts protons outward whereas AM2 does not[16] Consistent with these functional differences, solid-state NMR data indicate that His[19] in BM2 protonates with significantly lower proton-dissociation equilibrium constants (pKa’s) compared to His[37] in AM217 and to a peripheral His[27] in BM218. The micelle-bound BM2(1–33) structure shows a pore-facing Phe[515] whereas inter-helical distances in lipid bilayers indicate outward-facing phenylene rings[17]. We find that BM2-TM adopts relatively uniform α-helical torsion angles that are very similar to the AM2 TM conformation, suggesting that the proton-conduction differences between the two channels mainly result from the amino acid sidechains. The bilayer-bound BM2-TM has very different 15N chemical shifts as well as different helical length from micelle-bound BM2, indicating that the membrane environment exerts a significant influence on the conformation of this proton channel
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