Amantadine-induced conformational and dynamical changes of the influenza M2 transmembrane proton channel

Abstract

The M2 protein of influenza A virus forms a transmembrane proton channel important for viral infection and replication. Amantadine blocks this channel, thus inhibiting viral replication. Elucidating the high-resolution structure of the M2 protein and its change upon amantadine binding is crucial for designing antiviral drugs to combat the growing resistance of influenza A viruses against amantadine. We used magic-angle-spinning solid-state NMR to determine the conformation and dynamics of the transmembrane domain of the protein M2TMP in the apo- and amantadine-bound states in lipid bilayers. (13)C chemical shifts and torsion angles of the protein in 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC) bilayers indicate that M2TMP is alpha-helical in both states, but the average conformation differs subtly, especially at the G34-I35 linkage and V27 side chain. In the liquid-crystalline membrane, the complexed M2TMP shows dramatically narrower lines than the apo peptide. Analysis of the homogeneous and inhomogeneous line widths indicates that the apo-M2TMP undergoes significant microsecond-time scale motion, and amantadine binding alters the motional rates, causing line-narrowing. Amantadine also reduces the conformational heterogeneity of specific residues, including the G34/I35 pair and several side chains. Finally, amantadine causes the helical segment N-terminal to G34 to increase its tilt angle by 3 degrees , and the G34-I35 torsion angles cause a kink of 5 degrees in the amantadine-bound helix. These data indicate that amantadine affects the M2 proton channel mainly by changing the distribution and exchange rates among multiple low-energy conformations and only subtly alters the average conformation and orientation. Amantadine-resistant mutations thus may arise from binding-incompetent changes in the conformational equilibrium.

Fig. 1.

¹³C CP-MAS spectra of M2TMP at 303 K with (a and c) and without (b and d) amantadine. (a and b) LAGI. (c and d) VAIL. Note the significant line-narrowing and intensity increase in the presence of amantadine.

Fig. 2.

2D ¹³C¹³C DQ-filtered spectra of M2TMP in DLPC bilayers without (black) and with (red) amantadine at 243 K. Intraresidue connectivities and cross-peaks with chemical-shift changes are indicated. (a) LAGI. (b) VAIL. (c) Selected 1D cross-sections that exhibit line-narrowing and chemical-shift changes upon amantadine binding. The G34α trace was extracted from 1D CP spectra.

Fig. 3.

Amantadine-induced isotropic shift and T₂ changes of M2TMP. (a–c) Secondary shifts are plotted for C^α (a), C^β (b), and C′ (c). Open and filled bars correspond to the apo and complexed M2TMP, respectively. The average chemical-shift uncertainty is 0.35 ppm, estimated from the intrinsic line widths of the spectra. (d) Average absolute chemical-shift changes (filled squares) and fractional ¹³C T₂ increase at 303 K (open circles). Local maxima of chemical shift and T₂ perturbation occur at V27 and G34.

Fig. 4.

Selected torsion angle data of M2TMP without (open squares) and with (filled squares) amantadine, along with their rmsd-quantified best-fit simulations (lines). Thick lines indicate best-fit curves for the complexed M2TMP when different from the apo peptide. (a) V27 φ. (b) I35 φ. (c) G34 ψ. (Inset) The rmsd between the simulation and the experiment. (d) V27 χ_1H.

Fig. 5.

Orientation of amantadine-bound M2TMP. (a and b) ¹⁵N¹H dipolar coupling of unoriented M2TMP in DLPC bilayers with amantadine (filled squares, thick line). For comparison, the apo peptide data published recently are superimposed (open squares, thin line) (31). (a) V28. (b) A30. (c) PISA wheels of M2TMP constructed from the δ_// NH dipolar couplings and δ_⊥ ¹⁵N anisotropic shifts. The data fit to a wheel with a tilt angle τ of 38° (thick line). The apo peptide shows a τ = 35° (open symbols, thin line) (31).

Fig. 6.

Chemical shift and torsion-angle restrained backbone and partial side chain structure of amantadine-bound M2TMP. (a) Side view. (b) Top view. The exact position and orientation of amantadine is unknown and is shown here only as a reference to the peptide. The G34 ψ and I35 φ angles create a helix kink of 5°, highlighted by the blue N-terminal and the cyan C-terminal segments.