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

ABSTRACT: The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine. 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, 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, 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 A-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 (2)H 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.

SUBMITTER: Cady SD

PROVIDER: S-EPMC2818718 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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

Cady Sarah D SD Schmidt-Rohr Klaus K Wang Jun J Soto Cinque S CS Degrado William F WF Hong Mei M

Nature 20100201 7281

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine. 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, indicating a physical occlusion mechanism for inhibition. However, a solution NMR structure of M ...[more]

PMID: 20130653

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Project description:The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the side-chain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting structural findings from non-bilayer samples.

Dataset Information

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

Publications

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

Similar Datasets

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