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.

Figure 1. Drug-protein proximities from ¹³C{²H} REDOR spectra of Amt-bound M2 in DMPC bilayers at two Amt/P ratios

Control (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.

Figure 2. ²H NMR spectra of d₁₅-Amt in DMPC bilayers as a function of temperature and Amt/P

a. 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.

Figure 3. M2-Amt distance quantification

a-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.

Figure 4. SSNMR structure of Amt-bound M2 in lipid bilayers

a. Side view showing Ser 31, Val 27, Gly 34, His 37, Trp 41, and Amt in the high-affinity luminal site. Ser31 Cα lies in the mid-plane between the two rings of deuterons. The instantaneous orientation of Amt, which is slightly tilted from the channel axis, is shown. The time-averaged Amt orientation is parallel to the channel axis. b Top view showing the Ser 31 and Val 27 pore radii. This ribbon diagram was generated using the program Insight II.

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 M2

a. 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 . 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.