Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers

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Abstract

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine1. Resistance to these drugs has compromised their effectiveness against many influenza strains, including pandemic H1N1. A recent crystal structure of M2(22–46) showed electron densities attributed to a single amantadine in the amino-terminal half of the pore2, indicating a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18–60) showed four rimantadines bound to the carboxy-terminal lipid-facing surface of the helices3, suggesting an allosteric mechanism. Here we show by solid-state NMR spectroscopy that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine, is located in the N-terminal channel lumen, surrounded by residues mutated in amantadine-resistant viruses. Quantification of the protein–amantadine distances resulted in a 0.3 Å-resolution structure of the high-affinity binding site. The second, low-affinity, site was observed on the C-terminal protein surface, but only when the drug reaches high concentrations in the bilayer. The orientation and dynamics of the drug are distinct in the two sites, as shown by 2H NMR. These results indicate that amantadine physically occludes the M2 channel, thus paving the way for developing new antiviral drugs against influenza viruses. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes.

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Figure 1: Drug–protein proximities from 13 C{ 2 H} REDOR spectra of Amt-bound M2 in DMPC bilayers at two Amt/P ratios.
Figure 2: 2 H NMR spectra of d 15 -Amt in DMPC bilayers as a function of temperature and Amt/P.
Figure 3: M2–Amt distance quantification.
Figure 4: SSNMR structure of Amt-bound M2 in lipid bilayers.
Figure 5: Comparison of the high-pH SSNMR structure of Amt-bound M2 in lipid bilayers with the low-pH crystal structure of Amt-bound M2.

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Acknowledgements

This work was supported by a NSF grant MCB-0543473 and an NIH grant GM088204 to M.H., the Iowa State University Foundation, and NIH grants GM56423 and AI74571 to W.F.D.

Author Contributions S.D.C, M.H. and K.S-R. conducted SSNMR experiments. J.W. synthesized perdeuterated Amt and unlabeled M2. K.S-R. carried out distance simulations. S.D.C., M.H., C.S.S. and W.F.D. analysed the data and calculated the structure. M.H. and W.F.D. wrote the paper with inputs from other authors. M.H. designed and supervised the project.

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Correspondence to Mei Hong.

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Competing interests

W.F.D. chairs the scientific advisory board of InfluMedix, a company that is working on the pharmaceutical intervention of influenza virus infections.

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Cady, S., Schmidt-Rohr, K., Wang, J. et al. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463, 689–692 (2010) doi:10.1038/nature08722

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