Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain.
Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain.
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DOI：
10.1021/ja909968n
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发表时间：
2010-04-07
影响因子：
15
通讯作者：
Yang H
中科院分区：
文献类型：
--
作者：
Flynn EM;Hanson JA;Alber T;Yang H
结核分枝杆菌蛋白酪氨酸磷酸酶PtpB显示出对感染宿主巨噬细胞内普遍存在的氧化条件的抗性，但这种分子适应的机制尚不清楚。PtpB的晶体结构先前揭示了封闭的双螺旋盖覆盖活性位点。通过测量单分子Förster型共振能量转移来探测构成盖子的两个螺旋的动力学，我们获得了PtpB大的自发开放跃迁的直接证据，两个螺旋的闭合形式有利于~3：1。尽管构象相似的人口，两个螺旋的异步移动所证明的不同的开放和关闭率在我们的实验条件下。假设盖子关闭排除氧化剂，打开和关闭的速率定量地解释了观察到的氧化失活的缓慢速率。使用甘油而不是PEG 8000增加溶剂粘度导致更高的氧化失活率，这是由于开放构象异构体的数量增加。这些结果表明，快速构象门控的PtpB盖构成了可逆的物理封锁，暂时掩盖了活性位点和延缓氧化失活。
The Mycobacterium tuberculosis protein tyrosine phosphatase PtpB shows resistance to the oxidative conditions that prevail within an infected host macrophage, but the mechanism of this molecular adaptation is unknown. Crystal structures of PtpB revealed previously that a closed, two-helix lid covers the active site. By measuring single-molecule Förster-type resonance energy transfer to probe the dynamics of two helices that constitute the lid, we obtained direct evidence for large, spontaneous opening transitions of PtpB with the closed form of both helices favored ~3:1. Despite similar populations of conformers, the two helices move asynchronously as demonstrated by different opening and closing rates under our experimental conditions. Assuming that lid closure excludes oxidant, the rates of opening and closing quantitatively accounted for the slow observed rate of oxidative inactivation. Increasing solvent viscosity using glycerol but not PEG8000 resulted in higher rates of oxidative inactivation due to an increase in the population of open conformers. These results establish that the rapid conformational gating of the PtpB lid constitutes a reversible physical blockade that transiently masks the active site and retards oxidative inactivation.
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