Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from Influenza A virus

Abstract

Amantadine is known to block the M2 proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain blocked with amantadine, built using orientational constraints obtained from solid-state NMR polarization-inversion-spin-exchange-at-the-magic-angle experiments. The data indicates a kink in the monomer between two helical fragments having 20 degrees and 31 degrees tilt angles with respect to the membrane normal. This monomer structure is then used to construct a plausible model of the tetrameric amantadine-blocked M2 trans-membrane channel. The influence of amantadine binding through comparative cross polarization magic-angle spinning spectra was also observed. In addition, spectra are shown of the amantadine-resistant mutant, S31N, in the presence and absence of amantadine.

FIGURE 1

The structure of Amantadine and its analog Rimantadine.

FIGURE 2

Spectral comparison of the M2-TMD with (bottom) and without (top) amantadine. (A) CPMAS NMR spectra of ¹⁵N^δ1-His³⁷ M2-TMD in DMPC/DMPG liposomes at pH 8.8 and 277 K. Asterisks indicate spinning side bands. (B) Static ¹⁵N spectra of ¹⁵N-(L26, L36, L38, L40, L43) M2-TMD uniformly aligned in DMPC bilayers at pH 9 and 298 K. (C) PISEMA spectra for the samples used in panel B.

FIGURE 3

Spectral comparison of the M2-TMD S31N mutant with (bottom) and without (top) amantadine. (A) Static ¹⁵N spectra of ¹⁵N-(L26, L36, L38, L40, L43) M2-TMD uniformly aligned in DMPC bilayers at pH 9 and 298 K. (B) PISEMA spectra for the samples used in panel A.

FIGURE 4

PISEMA spectra for ¹⁵N labeled M2-TMD samples uniformly aligned in DMPC bilayers in the presence of 10 mM amantadine at pH 8.8 and 308 K. Each panel shows data from separate selective labelings: (A) isoleucines; (B) leucines; (C) glycine, alanines, valines; and (D) tryptophan. Panels E and F are the data from side chain ¹⁵N of W41 and H37, respectively.

FIGURE 5

(A) Two PISA wheels in the M2-TMD PISEMA spectrum. Experimental PISEMA resonances are connected with lines based on resonance assignments. The PISEMA resonances are fitted with two PISA wheels with tilt angles of 20° and 31°. Question marks indicate the missing data points and their hypothetical positions. (B) PISEMA wave simulations of the ¹⁵N M2-TMD anisotropic chemical shifts and ¹⁵N-¹H dipolar couplings. (C) PISA helix fitting of the M2-TMD/amantadine PISEMA data. Two helices are fitted with 20° (green) and 31° (magenta) tilt angles. The curve-fitting χ² values are 1.65 and 6.86 for the dipolar couplings and the anisotropic chemical shifts, respectively.

FIGURE 6

(A) Backbone stick structure of one M2-TMD subunit with amantadine (amantadine not shown) based on PISEMA data from residues 26–43. Plausible positions of residues His³⁷ and Trp⁴¹ side chains constrained by PISEMA side-chain data are also shown, but their positions are not unique. (B) Comparison between experimental PISEMA resonances (open squares) and the simulated PISEMA resonances (solid circles) of the refined M2-TMD/amantadine structure shown in panel A. The solid line is the PISEMA ellipse, which represents the range of possible values within the chemical shift and dipolar tensors. (C) The monomer structure with cylinders along the helical fragment axes emphasizing the 11° kink near Gly³⁴.

FIGURE 7

The energy landscape of the M2TMD homotetramer. Symmetric rigid body monomer rotations were performed about the membrane bilayer normal, here represented as the Z axis. The graph gives a relative measure of helix-helix interaction energy. The figures at the top of each region are representative conformations for the region, with arrows indicating the average tilt direction.

FIGURE 8

(A) Tetrameric model of M2-TMD backbone atoms with His³⁷ and Trp⁴¹ side chains in the presence of amantadine (amantadine not shown). The model exhibits C₄ symmetry and reflects a minimal helix-helix interaction of the monomer with a pore diameter of ∼10 Å. The interfacial region is less α-helical, which may be a result of amantadine interactions. (B) A top view of tetrameric complex with a representative radially symmetric positioning of amantadine (shown in orange with amino group pointing away). (C) The channel width between monomer backbone atoms as a function of membrane layer depth shows a widening near the C-terminus (bottom).