CHARACTERIZATION OF THE METAL BINDING SITES OF KMTR

KMTR 金属结合位点的表征

• 批准号: 8170256
• 负责人: STEFANO MANGANI
• 金额: $ 0.03万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170256
• 关键词: Affinity; Base Sequence; Binding; Cells; Cobalt; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; Databases; Electrophoretic Mobility Shift Assay; Elements; Family; Fluorescence Anisotropy; Funding; Genes; Genus Mycobacterium; Grant; Institution; Ions; Mediating; Metal Binding Site; Metalloproteins; Metals; Mycobacterium tuberculosis; Nickel; Protein Binding; Proteins; Repression; Repressor Proteins; Research; Research Personnel; Resources; Source; Toxic effect; Transcription Repressor/Corepressor; United States National Institutes of Health; genetic regulatory protein; in vivo; mutant; novel; pathogen; promoter; response; sensor

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 细胞含有调节蛋白来检测和响应必需金属的缺乏或过量，以维持足够的原子来满足金属蛋白的需求，从而避免毒性。 ArsR-SmtB 转录抑制子家族与编码参与过量金属外流和/或隔离的蛋白质的基因启动子相关。当抑制子与金属效应器结合且原子数量超过最佳细胞配额时，就会发生去抑制。 SmtB 介导的抑制可通过 Zn(II) 缓解，ZiaR 通过 Zn(II) 缓解，ArsR 通过 As(III)、Sb(III) 和 Bi(III) 缓解，CadC 通过 Cd(II)、Pb(II) 和 Bi 缓解(III)、CzrA、Co(II) 和 Zn(II) 等。显然，这些传感器可以区分体内不同的金属，但决定哪些无机元素引起反应的因素仍有待定义。一种新型 ArsR-SmtB 家族转录抑制因子 KmtR 已从分枝杆菌中鉴定出来。缺乏 kmtR 的结核分枝杆菌突变体表现出编码推导的 CDF 家族金属输出蛋白的 Rv2025c 表达升高。基本培养基中的镍和钴减轻了 cdf 和 kmtR 启动子的 KmtR 依赖性抑制。电泳迁移率变动分析和荧光各向异性显示纯化的 KmtR 与包含来自 cdf 和 kmtR 操纵子启动子的二联体对称区域的核苷酸序列结合。 KmtR 与钴一起孵育会抑制 DNA 复合物组装和金属-蛋白质结合。 KmtR 是继已发现的 NmtR 蛋白之后结核分枝杆菌中第二个镍和钴金属感应阻遏蛋白。两种金属感应阻遏蛋白的存在表明钴和镍离子在这种病原体中具有特殊意义。 KmtR 对镍和钴的亲和力比 NmtR 更紧密。该结果与完全培养基中 KmtR 而非 NmtR 检测到的这些金属的基础水平一致。数据库中列出了 1000 多个编码 ArsR-SmtB 相关蛋白的基因。