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-sn-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, similar to 280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved C-13-N-15, C-13-C-13, and H-1-N-15 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed N-15/C-13 labeling and with deuterated protein, MAS at omega(r)/2 pi = 60 kHz, omega(0H)/2 pi = 1000 MHz, and 11-1 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 angstrom for backbone heavy atoms and 1.1 A 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.

Published in:
Journal Of The American Chemical Society, 137, 47, 14877-14886
Washington, American Chemical Society

 Record created 2016-02-16, last modified 2018-03-17

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