Magic-angle-spinning NMR of the drug resistant S31N M2 proton transporter from influenza A

Abstract

We report chemical shift assignments of the drug-resistant S31N mutant of M2(18-60) determined using 3D magic-angle-spinning (MAS) NMR spectra acquired with a (15)N-(13)C ZF-TEDOR transfer followed by (13)C-(13)C mixing by RFDR. The MAS spectra reveal two sets of resonances, indicating that the tetramer assembles as a dimer of dimers, similar to the wild-type channel. Helicies from the two sets of chemical shifts are shown to be in close proximity at residue H37, and the assignments reveal a difference in the helix torsion angles, as predicted by TALOS+, for the key resistance residue N31. In contrast to wild-type M2(18-60), chemical shift changes are minimal upon addition of the inhibitor rimantadine, suggesting that the drug does not bind to S31N M2.

Figure 1

ZF-TEDOR spectra of S31N M2_18-60 are shown in the presence (blue) and absence (red) of the inhibitor Rmt. Spectra were recorded at a ¹H frequency of 900 MHz and demonstrate ~0.7 ppm and ~0.5 ppm ¹⁵N and ¹³C backbone linewidths, respectively. Labels correspond to N-Cα cross-peaks (right) and nitrogen i of N_i-C_i-1 cross-peaks (left) unless otherwise indicated. The sample temperature was ~30 °C, the spinning frequency was 20 kHz, and the TEDOR mixing time was 1.2 ms.

Figure 2

An aromatic-aromatic ¹³C correlation spectrum using 400 ms of proton driven spin diffusion mixing shows cross-peaks between the two sets of chemical shifts. The sample temperature was ~10 °C and the spinning frequency was 14.287 kHz. The inset shows an illustration of a C₂ symmetric channel composed of a dimer of dimers viewed along the C₂ axis with the four alpha helices at the corners. The observed intermolecular correlation between carbons δ2 and ε1′ is also depicted in the inset.

Figure 3

3D ZF-TEDOR-RFDR pulse sequence used for assignments. Narrow and broad bars represent pulses of 90 and 180 degree nutation respectively. Vertical dashed lines indicate rotor synchronization. The parameter n determines the total TEDOR mixing time, and the parameter m likewise determines the RFDR mixing, by extension of the respective pulse trains. The phase cycle was φ₁=(16×1)(16×3), φ₂=(16×4)(16×2), φ₃=2, φ₄=1, φ₅=13, φ₆=2244, φ₇=1133, φ₈=1111222233334444, φ_rec=4242 1313 2424 3131 2424 3131 4242 1313. The REDOR mixing in ZF-TEDOR used xy-4, and the RFDR mixing used xy-16 phase alternation. All other pulses had a phase of 1. The phases of the pulses after t₁ evolution and before t₂ evolution were incremented for phase sensitive detection. Due to time constraints, only the first 4 values of the phase cycle were used.

Figure 4

Slices are shown from the NCACX and NCOCX regions of a 3D ZF-TEDOR-RFDR spectrum used for sequential assignments. For P25, the NCδ transfer was also efficient (second panel). 1.2 ms of TEDOR mixing and 4.8 ms of RFDR mixing with 83 kHz pulses were used. The direct acquisition used a 6 μs dwell time and 3072 points (~18.4 ms), the indirect ¹³C dimension was acquired with a 50 μs dwell time and 180 complex points (9 ms), and the ¹⁵N dimension used a 150 μs dwell time and 74 complex points (11.1 ms). The carrier frequency was set to 100 ppm for ¹⁵N, and 89 ppm for ¹³C. The spectrum was acquired in ~5 days and provided useful NCACX and NCOCX connectivity information. The sample temperature was ~30 °C, and the spinning frequency was 20 kHz.