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

Cady SD, Schmidt-Rohr K, Wang J, Soto CS, Degrado WF, Hong M - Nature (2010)

Bottom Line: Quantification of the protein-amantadine distances resulted in a 0.3 A-resolution structure of the high-affinity binding site.The orientation and dynamics of the drug are distinct in the two sites, as shown by (2)H NMR.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Iowa State University, Ames, Iowa 50011 2, USA.

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.

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Comparison of the high-pH SSNMR structure of Amt-bound M2 in lipid bilayers with the low-pH crystal structure of Amt-bound M2a. Side view of the high-pH SSNMR structure, showing Amt to be enclosed by Val 27 at the top and His 37 at the bottom. b. Side view of the low-pH crystal structure 2. The helices are splayed far apart near the C-terminus. c. C-terminal view of the high-pH structure, showing a well-sequestered drug. d. C-terminal view of the low-pH structure, showing a more solvent-accessible drug. The figure was generated using the program PyMOL.
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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 M2a. Side view of the high-pH SSNMR structure, showing Amt to be enclosed by Val 27 at the top and His 37 at the bottom. b. Side view of the low-pH crystal structure 2. The helices are splayed far apart near the C-terminus. c. C-terminal view of the high-pH structure, showing a well-sequestered drug. d. C-terminal view of the low-pH structure, showing a more solvent-accessible drug. The figure was generated using the program PyMOL.

Mentions: The present SSNMR structure has significant differences from previous proposed structures of the Amt-M2 complex 2,3. While the drug location is very similar to that of the low-pH crystal structure, the shape of the binding site differs dramatically (3.4-Å Cα RMSD between the structures). In the crystal structure, the helices splay far apart near the C-terminus (Fig. 5b), to minimize electrostatic repulsions among the protonated His 37. In the high-pH SSNMR structure, the helices close off the bottom of the site, fully sequestering the drug and explaining the improved affinity at higher pH (Fig. 5a). The backbone of the SSNMR structure is more similar to the high-pH solution NMR structure, with comparable distances involving Val27 Cγ1, Ser31 Cα, and Gly34 Cα (Supplementary Fig. S5). Thus, the drug may have been present in the lumen in the solution NMR sample but not observable without isotopic labeling. Alternatively it might have been truly absent from the lumen due to reduced affinity to the micelle-bound and structurally plastic protein 23-25. The current high-resolution structure also revises an earlier SSNMR chemical-shift-constrained M2 model, where the lack of protein-drug distances resulted in a large N-terminal vestibule, which would yield a highly solvent-accessible low-affinity drug (Supplementary Fig. S6-S7).


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

Cady SD, Schmidt-Rohr K, Wang J, Soto CS, Degrado WF, Hong M - Nature (2010)

Comparison of the high-pH SSNMR structure of Amt-bound M2 in lipid bilayers with the low-pH crystal structure of Amt-bound M2a. Side view of the high-pH SSNMR structure, showing Amt to be enclosed by Val 27 at the top and His 37 at the bottom. b. Side view of the low-pH crystal structure 2. The helices are splayed far apart near the C-terminus. c. C-terminal view of the high-pH structure, showing a well-sequestered drug. d. C-terminal view of the low-pH structure, showing a more solvent-accessible drug. The figure was generated using the program PyMOL.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2818718&req=5

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 M2a. Side view of the high-pH SSNMR structure, showing Amt to be enclosed by Val 27 at the top and His 37 at the bottom. b. Side view of the low-pH crystal structure 2. The helices are splayed far apart near the C-terminus. c. C-terminal view of the high-pH structure, showing a well-sequestered drug. d. C-terminal view of the low-pH structure, showing a more solvent-accessible drug. The figure was generated using the program PyMOL.
Mentions: The present SSNMR structure has significant differences from previous proposed structures of the Amt-M2 complex 2,3. While the drug location is very similar to that of the low-pH crystal structure, the shape of the binding site differs dramatically (3.4-Å Cα RMSD between the structures). In the crystal structure, the helices splay far apart near the C-terminus (Fig. 5b), to minimize electrostatic repulsions among the protonated His 37. In the high-pH SSNMR structure, the helices close off the bottom of the site, fully sequestering the drug and explaining the improved affinity at higher pH (Fig. 5a). The backbone of the SSNMR structure is more similar to the high-pH solution NMR structure, with comparable distances involving Val27 Cγ1, Ser31 Cα, and Gly34 Cα (Supplementary Fig. S5). Thus, the drug may have been present in the lumen in the solution NMR sample but not observable without isotopic labeling. Alternatively it might have been truly absent from the lumen due to reduced affinity to the micelle-bound and structurally plastic protein 23-25. The current high-resolution structure also revises an earlier SSNMR chemical-shift-constrained M2 model, where the lack of protein-drug distances resulted in a large N-terminal vestibule, which would yield a highly solvent-accessible low-affinity drug (Supplementary Fig. S6-S7).

Bottom Line: Quantification of the protein-amantadine distances resulted in a 0.3 A-resolution structure of the high-affinity binding site.The orientation and dynamics of the drug are distinct in the two sites, as shown by (2)H NMR.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Iowa State University, Ames, Iowa 50011 2, USA.

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.

Show MeSH
Related in: MedlinePlus