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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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2H NMR spectra of d15-Amt in DMPC bilayers as a function of temperature and Amt/Pa. No M2. The calculated spectrum for 303 K reproduces the 1:3 frequency ratio and 4:1 intensity ratio of the two splittings. b Amt/P = 1: 4. The sum spectrum reproduces the 303 K spectrum by 1:9 combination of the lipid-bound 303 K spectrum and peptide-bound 283 K spectrum (not shown). c. Amt/P = 4 : 4. The sum spectrum uses a 1:3 combination of the M2-bound spectrum (II) and lipid-bound spectrum (I). d. Amt orientation in the M2 channel. e. One of the two possible Amt orientations in the lipid bilayer.
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Figure 2: 2H NMR spectra of d15-Amt in DMPC bilayers as a function of temperature and Amt/Pa. No M2. The calculated spectrum for 303 K reproduces the 1:3 frequency ratio and 4:1 intensity ratio of the two splittings. b Amt/P = 1: 4. The sum spectrum reproduces the 303 K spectrum by 1:9 combination of the lipid-bound 303 K spectrum and peptide-bound 283 K spectrum (not shown). c. Amt/P = 4 : 4. The sum spectrum uses a 1:3 combination of the M2-bound spectrum (II) and lipid-bound spectrum (I). d. Amt orientation in the M2 channel. e. One of the two possible Amt orientations in the lipid bilayer.

Mentions: 2H NMR provided exquisite details on the orientation and dynamics of amantadine, whose unique symmetry and rigidity simplify analysis. Amantadine is a rigid amphiphile with a polar amine and a hydrophobic adamantane centered around a 3-fold axis, ZM. Three axial C-D bonds are parallel to ZM while twelve equatorial C-D bonds are at 70° or 110° (θPM) from ZM (Fig. 2d). Amantadine partitions strongly into protein-free DMPC vesicles and exhibit 2H quadrupolar splittings of 36 and 123 kHz with a 4:1 intensity ratio at 243 K (Fig. 2a). These splittings indicate fast anisotropic rotation of the molecule around ZM, which scales the couplings from the rigid-limit value of 125 kHz by (3cos2θPM−1)/2, giving 40 kHz for the twelve equatorial bonds and 125 kHz for the three axial bonds. Wobbling of the ZM axis by ∼6° likely accounts for the additional motional averaging. As the temperature increased to 303 K, the couplings decreased twofold (18 and 58 kHz) while maintaining the same 1:3 frequency ratio and 4:1 intensity ratio. The ±0.46 scaling factor indicates Amt rotates rapidly around the normal (n⃗) of the liquid-crystalline bilayer in addition to its own axis, with ZM tilted by 37° or 80° from n⃗ (Fig. 2e) 16.


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)

2H NMR spectra of d15-Amt in DMPC bilayers as a function of temperature and Amt/Pa. No M2. The calculated spectrum for 303 K reproduces the 1:3 frequency ratio and 4:1 intensity ratio of the two splittings. b Amt/P = 1: 4. The sum spectrum reproduces the 303 K spectrum by 1:9 combination of the lipid-bound 303 K spectrum and peptide-bound 283 K spectrum (not shown). c. Amt/P = 4 : 4. The sum spectrum uses a 1:3 combination of the M2-bound spectrum (II) and lipid-bound spectrum (I). d. Amt orientation in the M2 channel. e. One of the two possible Amt orientations in the lipid bilayer.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2818718&req=5

Figure 2: 2H NMR spectra of d15-Amt in DMPC bilayers as a function of temperature and Amt/Pa. No M2. The calculated spectrum for 303 K reproduces the 1:3 frequency ratio and 4:1 intensity ratio of the two splittings. b Amt/P = 1: 4. The sum spectrum reproduces the 303 K spectrum by 1:9 combination of the lipid-bound 303 K spectrum and peptide-bound 283 K spectrum (not shown). c. Amt/P = 4 : 4. The sum spectrum uses a 1:3 combination of the M2-bound spectrum (II) and lipid-bound spectrum (I). d. Amt orientation in the M2 channel. e. One of the two possible Amt orientations in the lipid bilayer.
Mentions: 2H NMR provided exquisite details on the orientation and dynamics of amantadine, whose unique symmetry and rigidity simplify analysis. Amantadine is a rigid amphiphile with a polar amine and a hydrophobic adamantane centered around a 3-fold axis, ZM. Three axial C-D bonds are parallel to ZM while twelve equatorial C-D bonds are at 70° or 110° (θPM) from ZM (Fig. 2d). Amantadine partitions strongly into protein-free DMPC vesicles and exhibit 2H quadrupolar splittings of 36 and 123 kHz with a 4:1 intensity ratio at 243 K (Fig. 2a). These splittings indicate fast anisotropic rotation of the molecule around ZM, which scales the couplings from the rigid-limit value of 125 kHz by (3cos2θPM−1)/2, giving 40 kHz for the twelve equatorial bonds and 125 kHz for the three axial bonds. Wobbling of the ZM axis by ∼6° likely accounts for the additional motional averaging. As the temperature increased to 303 K, the couplings decreased twofold (18 and 58 kHz) while maintaining the same 1:3 frequency ratio and 4:1 intensity ratio. The ±0.46 scaling factor indicates Amt rotates rapidly around the normal (n⃗) of the liquid-crystalline bilayer in addition to its own axis, with ZM tilted by 37° or 80° from n⃗ (Fig. 2e) 16.

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