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

  • Cady S
  • Schmidt-Rohr K
  • Wang J
 et al. 
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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 rimantadine(1). 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 pore(2), 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 helices(3), 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 angstrom-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 H-2 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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Authors

  • Sarah D. Cady

  • Klaus Schmidt-Rohr

  • Jun Wang

  • Cinque S. Soto

  • William F. Degrado

  • Mei Hong

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