The chemical and dynamical influence of the anti-viral drug amantadine on the M2 proton channel transmembrane domain

Abstract

The M(2) proton channel plays a vital role in the life cycle of the influenza A virus. His(37), the key residue in the M(2) transmembrane domain (M(2)-TMD), plays a central role in the proton conductance mechanism. The anti-influenza drug, amantadine, inhibits the channel activity through binding to the pore of the M(2) channel. The nuclear spin relaxation data and polarization inversion spin exchange at the magic angle spectra show that both the polypeptide backbone and His(37) side chain are more constrained in the presence of amantadine. Using (15)N cross polarization magic-angle spinning NMR spectroscopy, the protonation of His(37) of M(2)-TMD in lipid bilayers was monitored in the absence and presence of amantadine as a function of pH. Binding amantadine lowers the His(37) pK(a) values by approximately three orders of magnitude compared with the first pK(a) of histidine in amantadine-free M(2)-TMD. Amantadine's influence on the His(37) chemical properties suggests a novel mechanism by which amantadine may inhibit proton conductance.

FIGURE 1

Chemical shift assignments associated with the chemical states of histidines in M₂-TMD. In the presence of amantadine, His -HisH⁺ dimerization does not occur. (N.O., not observed.)

FIGURE 2

¹⁵N solid-state NMR spectra of M₂-TMD in lipid bilayer environments with (bottom) and without (top) amantadine bound. (A) CPMAS NMR spectra of His³⁷ (¹⁵N^δ1) M₂-TMD in DMPC/DMPG (4:1 molar ratio) at pH 8.8. Spectra were obtained at 277 K with a spinning rate of 3 kHz. (B) PISEMA spectra of five-site ¹⁵N leucine labeled M₂-TMD at pH 9.0 in DMPC bilayers at 298 K.

FIGURE 3

¹⁵N PISEMA spectra of ¹⁵N^ɛ2 His³⁷ labeled M₂-TMD in aligned DMPC planar bilayers with amantadine bound at pH 9.0. The PISEMA spectra were acquired on a 400 MHz spectrometer at 298 K.

FIGURE 4

CPMAS NMR spectra of His³⁷ (¹⁵N^δ1) M₂-TMD with and without amantadine at different pH values. Spectra were obtained on a Bruker DMX-300 NMR spectrometer at 277 K with a spinning rate of 3 kHz. Signals at ∼100 ppm are from the natural abundance amides from the M₂-TMD backbone. Spinning side bands are marked with asterisks.

FIGURE 5

Curves for N_proton versus pH in the presence (dashed line) and absence (solid line) of 10 mM amantadine. N_proton is the number of released protons from the His³⁷ tetrad at a given pH value. At each pH value, N_proton was calculated on the basis of the ratio of M₀ for different histidine species (54). The N_proton versus pH data in the presence of amantadine were fitted assuming that there is a single kind of titratable histidine in the channel, while for the amantadine-free M₂-TMD multiple titration states must be considered and the fitting curve was adopted from Hu et al. (52) for the purpose of comparison. Both fitting curves were generated using the nonlinear regression fitting program in Origin 6 (Microcal Software, Northampton, MA).