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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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M2-Amt distance quantificationa-b.13C{2H} REDOR spectra of Amt-bound M2 (Amt/P = 4 : 4) obtained by the single-2H-pulse REDOR. a. SID-M2 at 4.2 ms mixing. b. LVAG-M2 at 6.1 ms mixing. c-f. REDOR simulations. c. Definition of the pore radius R and height difference Z from the center of Amt. d. Ser31 Cα simulation. e. Val27 Cγ1 simulation. f. Gly34 Cα simulation.
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Figure 3: M2-Amt distance quantificationa-b.13C{2H} REDOR spectra of Amt-bound M2 (Amt/P = 4 : 4) obtained by the single-2H-pulse REDOR. a. SID-M2 at 4.2 ms mixing. b. LVAG-M2 at 6.1 ms mixing. c-f. REDOR simulations. c. Definition of the pore radius R and height difference Z from the center of Amt. d. Ser31 Cα simulation. e. Val27 Cγ1 simulation. f. Gly34 Cα simulation.

Mentions: To determine the sub-angstrom resolution structure of the high-affinity binding site, we quantified the M2-Amt distances using an alternative REDOR experiment containing multiple 13C pulses and one 2H pulse, thus minimizing 2H pulse imperfections and yielding REDOR intensities closely following the universal curve 19 (Supplementary Fig. 3). The experiment yielded significantly faster REDOR dephasing (Fig. 3) while confirming the relative dephasing of different sites in Fig. 1.


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)

M2-Amt distance quantificationa-b.13C{2H} REDOR spectra of Amt-bound M2 (Amt/P = 4 : 4) obtained by the single-2H-pulse REDOR. a. SID-M2 at 4.2 ms mixing. b. LVAG-M2 at 6.1 ms mixing. c-f. REDOR simulations. c. Definition of the pore radius R and height difference Z from the center of Amt. d. Ser31 Cα simulation. e. Val27 Cγ1 simulation. f. Gly34 Cα simulation.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: M2-Amt distance quantificationa-b.13C{2H} REDOR spectra of Amt-bound M2 (Amt/P = 4 : 4) obtained by the single-2H-pulse REDOR. a. SID-M2 at 4.2 ms mixing. b. LVAG-M2 at 6.1 ms mixing. c-f. REDOR simulations. c. Definition of the pore radius R and height difference Z from the center of Amt. d. Ser31 Cα simulation. e. Val27 Cγ1 simulation. f. Gly34 Cα simulation.
Mentions: To determine the sub-angstrom resolution structure of the high-affinity binding site, we quantified the M2-Amt distances using an alternative REDOR experiment containing multiple 13C pulses and one 2H pulse, thus minimizing 2H pulse imperfections and yielding REDOR intensities closely following the universal curve 19 (Supplementary Fig. 3). The experiment yielded significantly faster REDOR dephasing (Fig. 3) while confirming the relative dephasing of different sites in Fig. 1.

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