Structure and Mechanism of the Influenza A M218-60 Dimer of Dimers.
Structure and Mechanism of the Influenza A M218-60 Dimer of Dimers.
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甲型流感 M218-60 二聚体的结构和机制。
DOI：
10.1021/jacs.5b04802
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发表时间：
2015-12-02
影响因子：
15
通讯作者：
Griffin RG
中科院分区：
文献类型：
--
作者：
Andreas LB;Reese M;Eddy MT;Gelev V;Ni QZ;Miller EA;Emsley L;Pintacuda G;Chou JJ;Griffin RG
我们报道了来自甲型流感病毒的M218-60的耐药S31N突变的魔角旋转（MAS）核磁共振结构。该蛋白分散在二phytanyl - sen -glycero-3-phosphocholine脂质双分子层中，光谱和广泛的约束条件表明M218-60由二聚体的二聚体组成。通过偶极子耦合实验，得到了~280个结构约束，得到了分辨良好的13C-15N、13C-13C和1H-15N的二维、三维和四维MAS光谱，这些光谱都显示出交叉峰加倍。螺旋间距离测量采用混合15N/13C标记和氘化蛋白，ωr/2π = 60 kHz, ω0H/2π = 1000 MHz的MAS， 1H检测甲基-甲基接触。实验揭示了由四个跨膜螺旋组成的四聚体的紧凑结构，其中两个相对的螺旋相对于四重对称排列在膜法线方向上移位和旋转，产生了双重对称结构。重要的门控和ph感应残基W41和H37的侧链构象与四重对称明显不同。骨架重原子的均方根偏差为0.7 Å，所有重原子的均方根偏差为1.1 Å。这种双重对称结构不同于以前所有的M2结构，其中许多是在洗涤剂和/或不完全活跃的较短结构中确定的。由于发现His和Trp残基在不同螺旋上的距离很短，因此该结构对H+输运机制具有重要意义。该结构在金刚烷基抑制剂结合位点附近也表现出双重对称，空间约束可能解释了耐药S31N突变的机制。
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 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 ωr/2π = 60 kHz, ω0H/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.