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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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Drug-protein proximities from 13C{2H} REDOR spectra of Amt-bound M2 in DMPC bilayers at two Amt/P ratiosControl (S0), dephased (S, red), and difference (ΔS) spectra at specified mixing times are shown. a. Ser31, Ile32, Asp44-labeled (SID) M2 at the stoichiometric ratio of Amt/P = 1 : 4. b. SID-M2 at the 4-fold excess ratio of Amt/P = 4 : 4. Ser31 Cα is dephased under both conditions but Asp44 Cα is dephased only when Amt is in excess. c-d. Leu26, Val27, Ala29, and Gly34-labeled (LVAG) M2 at Amt/P = 4 : 4. c. Gly34 Cα region. d. Val27 Cγ1 region.
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Figure 1: Drug-protein proximities from 13C{2H} REDOR spectra of Amt-bound M2 in DMPC bilayers at two Amt/P ratiosControl (S0), dephased (S, red), and difference (ΔS) spectra at specified mixing times are shown. a. Ser31, Ile32, Asp44-labeled (SID) M2 at the stoichiometric ratio of Amt/P = 1 : 4. b. SID-M2 at the 4-fold excess ratio of Amt/P = 4 : 4. Ser31 Cα is dephased under both conditions but Asp44 Cα is dephased only when Amt is in excess. c-d. Leu26, Val27, Ala29, and Gly34-labeled (LVAG) M2 at Amt/P = 4 : 4. c. Gly34 Cα region. d. Val27 Cγ1 region.

Mentions: To select for the highest-affinity binding site, we first measured the REDOR spectra of Amt-complexed M2 at an Amt/peptide molar ratio (Amt/P) of 1:4 (one drug per tetramer). At this stoichiometric concentration, Amt binds only to the luminal site: Fig. 1a shows 13C{2H} REDOR spectra without (S0) and with (S) multiple 2H dephasing pulses 14. The Ser31 Cα signal is strongly dephased by the deuterons (S/S0 = 0.76 ± 0.03 at 10.1 ms), indicating that Amt binds near Ser 31. In contrast, the Asp44 Cα signal in the peripheral site is unaffected. A double-quantum-filtered REDOR experiment that removed all lipid signals confirmed the lack of Asp 44 dephasing (Supplementary Fig. 2).


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)

Drug-protein proximities from 13C{2H} REDOR spectra of Amt-bound M2 in DMPC bilayers at two Amt/P ratiosControl (S0), dephased (S, red), and difference (ΔS) spectra at specified mixing times are shown. a. Ser31, Ile32, Asp44-labeled (SID) M2 at the stoichiometric ratio of Amt/P = 1 : 4. b. SID-M2 at the 4-fold excess ratio of Amt/P = 4 : 4. Ser31 Cα is dephased under both conditions but Asp44 Cα is dephased only when Amt is in excess. c-d. Leu26, Val27, Ala29, and Gly34-labeled (LVAG) M2 at Amt/P = 4 : 4. c. Gly34 Cα region. d. Val27 Cγ1 region.
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Related In: Results  -  Collection

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

Figure 1: Drug-protein proximities from 13C{2H} REDOR spectra of Amt-bound M2 in DMPC bilayers at two Amt/P ratiosControl (S0), dephased (S, red), and difference (ΔS) spectra at specified mixing times are shown. a. Ser31, Ile32, Asp44-labeled (SID) M2 at the stoichiometric ratio of Amt/P = 1 : 4. b. SID-M2 at the 4-fold excess ratio of Amt/P = 4 : 4. Ser31 Cα is dephased under both conditions but Asp44 Cα is dephased only when Amt is in excess. c-d. Leu26, Val27, Ala29, and Gly34-labeled (LVAG) M2 at Amt/P = 4 : 4. c. Gly34 Cα region. d. Val27 Cγ1 region.
Mentions: To select for the highest-affinity binding site, we first measured the REDOR spectra of Amt-complexed M2 at an Amt/peptide molar ratio (Amt/P) of 1:4 (one drug per tetramer). At this stoichiometric concentration, Amt binds only to the luminal site: Fig. 1a shows 13C{2H} REDOR spectra without (S0) and with (S) multiple 2H dephasing pulses 14. The Ser31 Cα signal is strongly dephased by the deuterons (S/S0 = 0.76 ± 0.03 at 10.1 ms), indicating that Amt binds near Ser 31. In contrast, the Asp44 Cα signal in the peripheral site is unaffected. A double-quantum-filtered REDOR experiment that removed all lipid signals confirmed the lack of Asp 44 dephasing (Supplementary Fig. 2).

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