Magic angle spinning NMR investigation of influenza A M2(18-60): support for an allosteric mechanism of inhibition

Abstract

The tetrameric M2 proton channel from influenza A virus conducts protons at low pH and is inhibited by aminoadamantyl drugs such as amantadine and rimantadine (Rmt). We report magic angle spinning NMR spectra of POPC and DPhPC membrane-embedded M2(18-60), both apo and in the presence of Rmt. Similar line widths in the spectra of apo and bound M2 indicate that Rmt does not have a significant impact on the dynamics or conformational heterogeneity of this construct. Substantial chemical shift changes for many residues in the transmembrane region support an allosteric mechanism of inhibition. An Rmt titration supports a binding stoichiometry of >1 Rmt molecule per channel and shows that nonspecific binding or changes in membrane composition are unlikely sources of the chemical shift changes. In addition, doubling of spectral lines in all of the observed samples provides evidence that the channel assembles with twofold symmetry.

Figure 1

(top) ¹⁵N-¹³C zf-TEDOR spectra (τ_mix= 1.3 ms) showing assignments of ¹³C,¹⁵N[¹²C,¹⁴N-ILFY]M2_18-60 in the drug bound (blue) and unbound (red) states. Unless otherwise indicated, cross peaks arise from 1-bond N-Cα magnetization transfer. (bottom) ¹³C-¹³C PDSD spectra (τ_mix= 50 ms) showing Asp and Gly cross-peaks of these samples. Sizable chemical shift changes are observed in the N and or Cα sites for residues 25, 27, 28, 31, 34, 35, 37, and 41. Many peaks are doubled (see supplemental Figure S1 for expansion), notably P25 and A29, supporting the existence of a twofold symmetric tetramer. Spectra were recorded ~0 °C, just above the phase transition of pure POPC lipids. Labels such as D44 are shown in italics to indicate less certainty in assignments (see Supplemental Information).

Figure 2

Chemical shift perturbations (Δδ = δ_bound−δ_apo) are distributed across the channel and support an allosteric effect upon drug binding. (left) Chemical shift perturbations as a function of residue number. (right) A comparison of the Rmt drug size with the transmembrane tetramer assembly from the solution structure. Blue residues indicate a shift of >2 ppm in N and or >1 ppm in Cα/Cβ. One of the four helices has been removed for clarity.

Figure 3

TEDOR spectra acquired at 0, 1, and 4 Rmt molecules per channel in a, b, and c, result in cross peaks due to M2 bound to Rmt and present at ~0%, 25% and >90%, respectively. The apo spectrum is simultaneously observed at 100%, 75% and <10% of total site-specific signal intensity. Unless otherwise indicated, cross peaks arise from 1-bond N-Cα magnetization transfer. Resonances that clearly show the titration are displayed in red (unbound form) and blue (Rmt bound resonances). Dashed lines at G34 and other resonances serve as a guide. Signal to noise is ~10 for strong signals. M2 samples used in the titration were embedded in DPhPC lipids and show nearly identical spectra as those recorded in POPC lipids (see Figure 4).

Figure 4

¹³C-¹³C PDSD spectra (τ_mix= 15 ms) of POPC embedded (red) and DPhPC embedded (black) M2 show nearly identical spectra with maximum chemical shift differences of 0.3 ppm. With 15 ms mixing, mostly 1-bond correlations are observed.