Mechanism of metal ion activation of the diphtheria toxin repressor DtxR
Mechanism of metal ion activation of the diphtheria toxin repressor DtxR
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DOI：
10.1073/pnas.0500908102
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发表时间：
2005-12-20
影响因子：
11.1
通讯作者：
Ringe, D
中科院分区：
文献类型：
--
作者：
D'Aquino, JA;Tetenbaum-Novatt, J;Ringe, D
白喉毒素抑制因子（DtxR）是一种金属离子激活的转录调节因子，与白喉棒状杆菌的毒力有关。结构测定表明，每个阻遏物单体有两个金属离子结合位点，位点定向诱变表明，结合位点2（主要）对于识别目标DNA阻遏物至关重要，而结合位点1（辅助）的作用尚不清楚。量热技术表明，虽然结合位点1（辅助）对金属离子具有高亲和力，结合常数为2 × 10(-7)，但结合位点2（主要）是一个低亲和力的结合位点，结合常数为6.3 × 10（-4）。这两个结合位点以独立的方式起作用，它们的作用可以很容易地通过传统的突变分析来分析。我们的研究结果清楚地表明，在金属离子激活过程中，结合位点1（辅助）是第一个被占据的，在抑制因子的稳定中起着关键作用。此外，本文报道的突变体Ni-DtxR（H79A,C102D）和之前报道的DtxR（H79A）的结构数据使我们能够提出金属活化DtxR的机制。
The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10(-7), binding site 2 (primary) is a low-affinity binding site with a binding constant of 6.3 x 10(-4). These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.