Department of Biology, University of Maryland College Park, College Park, Maryland, USA.
Nat Struct Mol Biol. 2010 Apr;17(4):451-8. doi: 10.1038/nsmb.1775. Epub 2010 Mar 7.
Under prolonged stimulation, the mechanosensitive channel MscS of Escherichia coli enters a tension-insensitive inactivated state. We transformed the delipidated crystal structure and restored the link between lipid-facing TM1 and TM2 and gate-forming TM3 helices. Joining the conserved Phe68 of TM2 with Leu111 of TM1, this buried contact mediated opening in steered molecular dynamics simulations with forces applied to the peripheral helices. Both F68S and L111S substitutions produced severe loss-of-function phenotypes in vivo by increasing the inactivation rate and promoting unusual 'silent' inactivation from the resting state. F68S also suppressed the noninactivating phenotype of G113A. The L111C cysteine buried in the TM2-TM3 crevice was accessible to methanethiosulfonate-ethyltrimethylammonium (MTSET) only in the inactivated state, which was stabilized upon modification by a positive charge. The restored interhelical contact thus is critically involved in force transmission from the lipid-facing helices to the gate, and inactivation appears to be a result of TM2-TM3 uncoupling.
在长时间的刺激下，大肠杆菌的机械敏感通道 MscS 进入张力不敏感的失活状态。我们转化了去脂的晶体结构，并恢复了脂质面向的 TM1 和 TM2 以及形成门的 TM3 螺旋之间的连接。通过将 TM2 上的保守苯丙氨酸 68 与 TM1 上的亮氨酸 111 连接起来，在力施加到外周螺旋的引导分子动力学模拟中，这种埋藏的接触介导了开口。F68S 和 L111S 取代均通过增加失活率和促进来自静止状态的异常“沉默”失活，在体内产生严重的功能丧失表型。F68S 还抑制了 G113A 的非失活表型。位于 TM2-TM3 裂缝中的埋藏 L111C 半胱氨酸仅在失活状态下可被甲硫基三磺酸钠-乙基三甲基铵 (MTSET) 接近，修饰后带正电荷会使其稳定。因此，恢复的螺旋间接触对于从脂质面向螺旋向门的力传递至关重要，失活似乎是 TM2-TM3 解偶联的结果。
