The closed state of a H+ channel helical bundle combining precise orientational and distance restraints from solid state NMR-1
The closed state of a H+ channel helical bundle combining precise orientational and distance restraints from solid state NMR-1
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DOI：
10.1021/bi0262799
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发表时间：
2002-11-05
期刊：
影响因子：
2.9
通讯作者：
Cross, TA
中科院分区：
文献类型：
--
作者：
Nishimura, K;Kim, SG;Cross, TA
通过 REDOR 固态核磁共振波谱法，从甲型流感病毒外壳的 M2 质子通道中精确测量了 M2-TMP 跨膜四聚束中的螺旋间距离。螺旋主链的高分辨率结构已通过水合二肉豆蔻酰磷脂酰胆碱双层中均匀排列的肽制剂的方向限制来确定。此处，N-15(pi) 标记的 His37 和 C-13(gamma) 标记的 Trp41 之间的距离被确定为小于 3.9 A。如此短的距离，与已知的各个螺旋的倾斜和旋转方向相结合，不仅可以确定哪些特定侧链配对引起相互作用，而且还可以表征四聚体束的侧链扭转角度和约束。由此产生的质子通道结构通过多种方式得到验证。组氨酸和色氨酸侧链都朝向孔，在那里它们可以发挥重要的功能作用。该通道似乎因四个吲哚的接近而关闭，这与 pH 7.0 及以上完整蛋白质的电生理学和诱变研究一致。该孔保持了膜 N 端侧的完整性，同时，生成了一个足以结合金刚烷胺的空腔。最后，在 (15)N(pi)His37 的 PISEMA 谱中观察到 2 kHz 耦合，根据观察到的 REDOR 距离验证了 His37 侧链的方向。
An interhelical distance has been precisely measured by REDOR solid-state NMR spectroscopy in the transmembrane tetrameric bundle of M2-TMP, from the M2 proton channel of the influenza A viral coat. The high-resolution structure of the helical backbone has been determined using orientational restraints from uniformly aligned peptide preparations in hydrated dimyristoylphosphatidylcholine bilayers. Here, the distance between N-15(pi) labeled His37 and C-13(gamma) labeled Trp41 is determined to be less than 3.9 A. Such a short distance, in combination with the known tilt and rotational orientation of the individual helices, permits not only a determination of which specific side chain pairings give rise to the interaction, but also the side chain torsion angles and restraints for the tetrameric bundle can also be characterized. The resulting proton channel structure is validated in a variety of ways. Both histidine and tryptophan side chains are oriented in toward the pore where they can play a significant functional role. The channel appears to be closed by the proximity of the four indoles consistent with electrophysiology and mutagenesis studies of the intact protein at pH 7.0 and above. The pore maintains its integrity to the N terminal side of the membrane, and at the same time, a cavity is generated that appears adequate for binding amantadine. Finally, the observation of a 2 kHz coupling in the PISEMA spectrum of (15)N(pi)His37 validates the orientation of the His37 side chain based on the observed REDOR distance.