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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Cady, S. D., Luo, W. B., Hu, F. & Hong, M. Structure and function of the influenza M2 proton channel. Biochemistry 48, 7356–7364 (2009)
Stouffer, A. L. et al. Structural basis for the function and inhibition of an influenza virus proton channel. Nature 451, 596–599 (2008)
Schnell, J. R. & Chou, J. J. Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, 591–595 (2008)
Pinto, L. H. & Lamb, R. A. The M2 proton channels of influenza A and B viruses. J. Biol. Chem. 281, 8997–9000 (2006)
Salom, D., Hill, B. R., Lear, J. D. & DeGrado, W. F. pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus. Biochemistry 39, 14160–14170 (2000)
Luo, W. & Hong, M. Determination of the oligomeric number and intermolecular distances of membrane protein assemblies by anisotropic 1H-driven spin diffusion NMR spectroscopy. J. Am. Chem. Soc. 128, 7242–7251 (2006)
Stouffer, A. L. et al. The interplay of functional tuning, drug resistance, and thermodynamic stability in the evolution of the M2 proton channel from the influenza A virus. Structure 16, 1067–1076 (2008)
Ma, C. et al. Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel. Proc. Natl Acad. Sci. USA 106, 12283–12288 (2009)
Wang, C., Takeuchi, K., Pinto, L. H. & Lamb, R. A. Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block. J. Virol. 67, 5585–5594 (1993)
Jing, X. et al. Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel. Proc. Natl Acad. Sci. USA 105, 10967–10972 (2008)
Holsinger, L. J., Nichani, D., Pinto, L. H. & Lamb, R. A. Influenza A virus M2 ion channel protein: a structure-function analysis. J. Virol. 68, 1551–1563 (1994)
Gullion, T. & Schaefer, J. Rotational echo double resonance NMR. J. Magn. Reson. 81, 196–200 (1989)
Luo, W., Mani, R. & Hong, M. Sidechain conformation and gating of the M2 transmembrane peptide proton channel of influenza A virus from solid-state NMR. J. Phys. Chem. 111, 10825–10832 (2007)
Jaroniec, C. P., Tounge, B. A., Herzfeld, J. & Griffin, R. G. Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate 13C–15N distance measurements in uniformly 13C, 15N-labeled peptides. J. Am. Chem. Soc. 123, 3507–3519 (2001)
Cady, S. D., Mishanina, T. V. & Hong, M. Structure of amantadine-bound M2 transmembrane peptide of influenza A in lipid bilayers from magic-angle-spinning solid-state NMR: the role of Ser31 in amantadine binding. J. Mol. Biol. 385, 1127–1141 (2009)
Li, C., Yi, M., Hu, J., Zhou, H. X. & Cross, T. A. Solid-state NMR and MD simulations of the antiviral drug amantadine solubilized in DMPC bilayers. Biophys. J. 94, 1295–1302 (2008)
Yi, M., Cross, T. A. & Zhou, H. X. A secondary gate as a mechanism for inhibition of the M2 proton channel by amantadine. J. Phys. Chem. B 112, 7977–7979 (2008)
Chen, H., Wu, Y. & Voth, G. A. Proton transport behavior through the influenza A M2 channel: insights from molecular simulation. Biophys. J. 93, 3470–3479 (2007)
Gullion, T. Measuring 13C–2D dipolar couplings with a universal REDOR dephasing curve. J. Magn. Reson. 146, 220–222 (2000)
Hu, J. et al. Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from influenza A virus. Biophys. J. 92, 4335–4343 (2007)
Tang, Y., Zaitseva, F., Lamb, R. A. & Pinto, L. H. The gate of the influenza virus M2 proton channel is formed by a single tryptophan residue. J. Biol. Chem. 277, 39880–39886 (2002)
Hu, J., Riqiang, F. & Cross, T. A. The chemical and dynamical influence of the anti-viral drug amantadine on the M2 proton channel transmembrane domain. Biophys. J. 93, 276–283 (2007)
Cristian, L., Lear, J. D. & DeGrado, W. F. Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers. Proc. Natl Acad. Sci. USA 100, 14772–14777 (2003)
Li, C., Qin, H., Gao, F. P. & Cross, T. A. Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel. Biochim. Biophys. Acta 1768, 3162–3170 (2007)
Cady, S. D. & Hong, M. Amantadine-induced conformational and dynamical changes of the influenza M2 transmembrane proton channel. Proc. Natl Acad. Sci. USA 105, 1483–1488 (2008)
Lin, T. I., Heider, H. & Schroeder, C. Different modes of inhibition by adamantane amine derivatives and natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein. J. Gen. Virol. 78, 767–774 (1997)
Wang, J. et al. Discovery of spiro-piperidine inhibitors and their modulation of the dynamics of the M2 proton channel from influenza A virus. J. Am. Chem. Soc. 131, 8066–8076 (2009)
Luo, W., Cady, S. D. & Hong, M. Immobilization of the influenza A M2 transmembrane peptide in virus–envelope mimetic lipid membranes: a solid-state NMR investigation. Biochemistry 48, 6361–6368 (2009)
Hong, M., Mishanina, T. V. & Cady, S. D. Accurate measurement of methyl 13C chemical shifts by solid-state NMR for the determination of protein sidechain conformation: the influenza M2 transmembrane peptide as an example. J. Am. Chem. Soc. 131, 7806–7816 (2009)
Sack, I., Goldbourt, A., Vega, S. & Buntkowsky, G. Deuterium REDOR: principles and applications for distance measurements. J. Magn. Reson. 138, 54–65 (1999)
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.
W.F.D. chairs the scientific advisory board of InfluMedix, a company that is working on the pharmaceutical intervention of influenza virus infections.
About this article
Cite this article
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
Review of Scientific Instruments (2019)
The L46P Mutant Confers a Novel Allosteric Mechanism of Resistance Toward the Influenza A Virus M2 S31N Proton Channel Blockers
Molecular Pharmacology (2019)
The Journal of Physical Chemistry B (2019)
Frontiers in Cellular and Infection Microbiology (2019)