A distal tyrosine residue is required for ligand discrimination in DevS from Mycobacterium tuberculosis.
A distal tyrosine residue is required for ligand discrimination in DevS from Mycobacterium tuberculosis.
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DOI：
10.1021/bi801234w
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发表时间：
2008-11-25
期刊：
影响因子：
2.9
通讯作者：
Moenne-Loccoz, Pierre
中科院分区：
文献类型：
--
作者：
Yukl, Erik T.;Ioanoviciu, Alexandra;Nakano, Michiko M.;de Montellano, Paul R. Ortiz;Moenne-Loccoz, Pierre
DEVS是一种以血红素为基础的传感器激酶，需要感知环境条件，从而导致结核分枝杆菌的非复制性持久性。当血红素是铁的5配位的高自旋或6配位的低自旋的CO或NO络合物时，可以观察到激酶活性，但在氧络合物中受到强烈的抑制。这些外源配体之间的区别被认为依赖于与结合氧的特定氢键网络。在这里，我们报道了野生型和Y171F DEVS在不同的血红素复合体中的共振拉曼数据和自磷酸化分析。Y171F突变消除了对CO、NO和O2的配体歧视，导致同样无效的复合体。相反，亚铁脱氧的Y171F变异株表现出与野生型相同的自激酶活性。Y171F氧基络合物的拉曼光谱表明，氧基的环境与野生型相比发生了显著的变化。他们还提出，远端口袋中的溶剂分子取代了Tyr羟基，成为氧基较差的氢键供体。野生型CO和NO复合体是CO或NO基团没有氢键的主要基团，而Y171F突变导致远端口袋极性略有增加。Y171F突变对血红素的近端环境没有影响，5配位亚铁脱氧野生型的活性在Y171F突变中是保守的。因此，虽然在亚铁-脱氧蛋白中缺乏外源配体导致适度的激酶活性，但Tyr171与远端双原子配体之间的相互作用会打开和关闭该激酶的活性。Y171F突变扰乱了开/关开关，并使远端配体处于不活跃状态。这一机制模型与Tyr171是识别远端配体所必需的是一致的，但对于自动磷酸化活性不是必需的。
DevS is a heme-based sensor kinase required for sensing environmental conditions leading to non-replicating persistence in M. tuberculosis. Kinase activity is observed when the heme is ferrous 5-coordinate high-spin or 6-coordinate low-spin CO or NO complex, but is strongly inhibited in the oxy complex. Discrimination between these exogenous ligands has been proposed to depend on a specific hydrogen bond network with bound oxygen. Here we report resonance Raman data and autophosphorylation assays of wild-type and Y171F DevS in various heme complexes. The Y171F mutation eliminates ligand discrimination for CO, NO and O2, resulting in equally inactive complexes. In contrast, the ferrous-deoxy Y171F variant exhibits equivalent autokinase activity as the wild-type. Raman spectra of the oxy complex of Y171F indicate that the environment of the oxy group is significantly altered from that in the wild-type. They also suggest that a solvent molecule in the distal pocket substitutes for the Tyr hydroxyl group to act as a poorer hydrogen bond donor to the oxy group. The wild-type CO and NO complexes exist as a major population in which the CO or NO groups are free of hydrogen bonds while the Y171F mutation results in a mild increase in the distal pocket polarity. The Y171F mutation has no impact on the proximal environment of the heme, and the activity observed with the 5-coordinate ferrous-deoxy wild-type is conserved in the Y171F variant. Thus, while the absence of an exogenous ligand in the ferrous-deoxy proteins leads to a moderate kinase activity, interactions between Tyr171 and distal diatomic ligands turn the kinase activity ON and OFF. The Y171F mutation disrupts the ON/OFF switch and renders all states with a distal ligand inactive. This mechanistic model is consistent with Tyr171 being required for distal ligand discrimination, but non-essential for autophosphorylation activity.
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