Magic-Angle-Spinning NMR of the Drug Resistant S31N M2 Proton Transporter from Influenza A

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 ¹⁵N–¹³C ZF-TEDOR transfer followed by ¹³C–¹³C 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

Structure and Mechanism of the Influenza A M2_18–60 Dimer of Dimers

We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M2_18–60 from Influenza A. The protein was dispersed in diphytanoyl-<i>sn</i>-glycero-3-phosphocholine lipid bilayers, and the spectra and an extensive set of constraints indicate that M2_18–60 consists of a dimer of dimers. In particular, ∼280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved ¹³C–¹⁵N, ¹³C–¹³C, and ¹H–¹⁵N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed ¹⁵N/¹³C labeling and with deuterated protein, MAS at ω_r/2π = 60 kHz, ω_0H/2π = 1000 MHz, and ¹H detection of methyl–methyl contacts. The experiments reveal a compact structure consisting of a tetramer composed of four transmembrane helices, in which two opposing helices are displaced and rotated in the direction of the membrane normal relative to a four-fold symmetric arrangement, yielding a two-fold symmetric structure. Side chain conformations of the important gating and pH-sensing residues W41 and H37 are found to differ markedly from four-fold symmetry. The rmsd of the structure is 0.7 Å for backbone heavy atoms and 1.1 Å for all heavy atoms. This two-fold symmetric structure is different from all of the previous structures of M2, many of which were determined in detergent and/or with shorter constructs that are not fully active. The structure has implications for the mechanism of H⁺ transport since the distance between His and Trp residues on different helices is found to be short. The structure also exhibits two-fold symmetry in the vicinity of the binding site of adamantyl inhibitors, and steric constraints may explain the mechanism of the drug-resistant S31N mutation