title: Structure and Mechanism of the Influenza A M218-60 Dimer of Dimers.
abstract: We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M218-60 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 M218-60 consists of a dimer of dimers. In particular, ∼280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved (13)C-(15)N, (13)C-(13)C, and (1)H-(15)N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed (15)N/(13)C labeling and with deuterated protein, MAS at ωr/2π = 60 kHz, ω0H/2π = 1000 MHz, and (1)H 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.
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Loren B
authors.initials: LB
authors.lastname: Andreas
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Marcel
authors.initials: M
authors.lastname: Reese
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Eddy
authors.affiliation: Department of Chemistry and Pharmacy, Sofia University , 1 James Bourchier Boulevard, 1164 Sofia, Bulgaria.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Gelev
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Qing Zhe
authors.initials: QZ
authors.lastname: Ni
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Miller
authors.affiliation: Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL) , 1015 Lausanne, Switzerland.
authors.forename: Lyndon
authors.initials: L
authors.lastname: Emsley
authors.affiliation: CNRS/ENS Lyon/UCB-Lyon 1, Université de Lyon , Centre RMN à Très Hauts Champs, 5 rue de la Doua, 69100 Villeurbanne, France.
authors.forename: Guido
authors.initials: G
authors.lastname: Pintacuda
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , Boston, Massachusetts 02115, United States.
authors.forename: James J
authors.initials: JJ
authors.lastname: Chou
authors.affiliation: Department of Chemistry and Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Griffin
mesh_terms: D019281:Dimerization; D008051:Lipid Bilayers; D011487:Protein Conformation; D014763:Viral Matrix Proteins / Q000737:chemistry*; D000086082:Viroporin Proteins
chemical_list: D008051:Lipid Bilayers; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins