Giuseppe
Emeritus
Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers (Nature, edited)
Letter
Nature 463, 689-692 (4 February 2010) | doi:10.1038/nature08722; Received 31 August 2009; Accepted 27 November 2009
Letter
Nature 463, 689-692 (4 February 2010) | doi:10.1038/nature08722; Received 31 August 2009; Accepted 27 November 2009
Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers
Sarah D. Cady1, Klaus Schmidt-Rohr1, Jun Wang2, Cinque S. Soto2, William F. DeGrado2 & Mei Hong1
1. Department of Chemistry, Iowa State University, Ames, Iowa 50011 2, USA
2. Department of Biochemistry & Biophysics, School of Medicine, and Department of Chemistry University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059, USA
Correspondence to: Mei Hong1
Correspondence and requests for materials should be addressed to M.H. (Email: mhong@iastate.edu).
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 ?-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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<cite cite="http://www.nature.com/nature/journal/v463/n7281/full/nature08722.html">Access : Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers : Nature</cite>Sarah D. Cady1, Klaus Schmidt-Rohr1, Jun Wang2, Cinque S. Soto2, William F. DeGrado2 & Mei Hong1
1. Department of Chemistry, Iowa State University, Ames, Iowa 50011 2, USA
2. Department of Biochemistry & Biophysics, School of Medicine, and Department of Chemistry University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059, USA
Correspondence to: Mei Hong1
Correspondence and requests for materials should be addressed to M.H. (Email: mhong@iastate.edu).
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 ?-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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