CHARACTERIZATION OF THE METAL BINDING SITES OF KMTR

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞内含有调节蛋白，以检测和响应必需金属的缺乏或过剩，以维持足够的原子，以满足金属蛋白的要求，避免毒性。ArsR-SmtB转录抑制物家族与编码蛋白的基因启动子有关，这些基因参与了过剩金属的外流和/或封存。当抑制物结合的金属效应器与超过最佳细胞配额的原子数量一致时，就会发生去抑制。SmtB介导的抑制作用可被锌(II)缓解，Ziar可被锌(II)缓解，ArsR可被As(III)、Sb(III)和Bi(III)缓解，CADC可被Cd(II)、Pb(II)和Bi(III)缓解，CHARA可被Co(II)和Zn(II)等缓解。显然，这些传感器在体内区分不同的金属，但决定哪些无机元素能引起反应的因素仍有待确定。从分枝杆菌中鉴定出一个新的ArsR-SmtB家族转录抑制因子KMTR。缺乏KMTR的结核分枝杆菌突变株显示编码CDF家族金属出口蛋白的Rv2025c表达增加。微量的镍和钴可减轻依赖KMTR的CDF和KMTR操纵子启动子的抑制作用。电泳迁移率变化分析和荧光各向异性分析表明，纯化的KMTR与来自CDF和KMTR操纵子-启动子的含有二联体对称区的核苷酸序列结合。KMTR与钴孵育可抑制DNA复合体组装和金属-蛋白质结合。KMTR是继已发现的NmtR蛋白之后，结核分枝杆菌对镍和钴的第二个金属感应抑制蛋白。两个金属感应抑制蛋白的存在表明钴和镍离子在该病中具有特殊的意义。与NMTR相比，KMTR对镍和钴的亲和力更强。这一结果与KMTR检测到的这些金属的基础水平是一致的，但在全介质中NMTR没有检测到。数据库中列出了1000多个编码ArsR-SmtB相关蛋白的基因。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cells contain regulatory proteins to detect and respond to deficiency or excess of essential metals to maintain sufficient atoms to satisfy the requirements of metalloproteins avoiding toxicity. The ArsR-SmtB family of transcriptional repressors associate with the promoters of genes encoding proteins involved in the efflux and/or sequestration of excess metal. De-repression occurs when the repressors bind metal effectors coincident with the number of atoms exceeding an optimal cell quota. SmtB-mediated repression is alleviated by Zn(II), ZiaR by Zn(II), ArsR by As(III), Sb(III), and Bi(III), CadC by Cd(II), Pb(II) and Bi(III), and CzrA by Co(II) and Zn(II) and many others. Clearly these sensors discriminate between different metals in vivo, but the factors dictating which inorganic elements elicit responses remain to be defined. A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterized from mycobacteria. Mutants of Mycobacterium tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays and fluorescence anisotropy show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. Incubation of KmtR with cobalt inhibits DNA complex assembly and metal-protein binding was also confirmed. KmtR was the second metal sensing repressor protein for nickel and cobalt in M. tuberculosis after the already discovered NmtR protein. The presence of two metal sensing repressor proteins suggesting a special significance of cobalt and nickel ions in this pathogen. KmtR has tighter affinities for nickel and cobalt than NmtR. This result is consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB-related proteins are listed in databases.