A nickel-cobalt-sensing ArsR-SmtB family repressor - Contributions of cytosol and effector binding sites to metal selectivity
A nickel-cobalt-sensing ArsR-SmtB family repressor - Contributions of cytosol and effector binding sites to metal selectivity
复制标题
DOI：
10.1074/jbc.m207677200
复制
发表时间：
2002-10-11
影响因子：
4.8
通讯作者：
Robinson, NJ
中科院分区：
文献类型：
--
作者：
Cave, JS;Meng, WM;Robinson, NJ
结核分枝杆菌NmtR是金属感受器转录抑制因子ArsR-SmtB家族的新成员。NmtR与编码P-1型ATPase(NMTA)的基因的操纵子-启动子结合，在体内抑制转录，除非在添加镍或某种程度上添加钴的介质中。在蓝藻宿主聚球藻PCC7942菌株R2-PIM8(SMT)中，NmtR介导的抑制可被钴而不是镍或锌的加入而减轻，而相关的传感器SmtB仅对锌有反应。对每个细胞镍原子数量的量化表明，NmtR对镍的敏感性与细胞内镍含量相关。SmtB(锌)和NmtR(钴)在普通胞液中对金属的区别不能简单地用平衡亲和力来解释；尽管NmtR确实比SmtB与镍结合得更紧密，但它对锌的亲和力比对钴的亲和力更高，对钴的结合比SmtB更弱。已知SmtB在C-末端α5螺旋上的螺旋间四配位四面体位置结合和传感锌，而Co(II)和Ni(II)取代的NmtR的吸收光谱显示五配位和六配位的金属络合物。定点突变鉴定了6个潜在的钴/镍配体，它们是诱导剂识别所必需的，但不受NmtR的抑制，其中4个(Asp(91)，His(93)，His(104)，His(107))与SmtB的α5配体结合，另外两个His由羧基末端的“延伸”(标为α5C)提供。凝胶滞留分析表明，在低亲和力钴的浓度下，锌并不以变构方式调节NmtR-DNA结合。这些数据表明，另外两个配体形成了六配位的金属络合物，对于推动NmtR对DNA结合的变构调节至关重要，从而使NmtR能够优先感觉到相对于SmtB更高配位数的金属。
NmtR from Mycobacterium tuberculosis is a new member of the ArsR-SmtB family of metal sensor transcriptional repressors. NmtR binds to the operator-promoter of a gene encoding a P-1 type ATPase (NmtA), repressing transcription in vivo except in medium supplemented with nickel or, to some extent, cobalt. In a cyanobacterial host, Synechococcus PCC 7942 strain R2-PIM8(smt), NmtR-mediated repression is alleviated by cobalt but not nickel or zinc addition, while the related sensor SmtB responds exclusively to zinc. Quantification of the number of atoms of nickel per cell shows that NmtR nickel sensitivity correlates with cytosolic nickel contents. Differential metal discrimination in a common cytosol by SmtB (zinc) and NmtR (cobalt) is not simply explained by affinities at equilibrium; although NmtR does bind nickel substantially more tightly than SmtB, it has a higher affinity for zinc than for cobalt and binds cobalt more weakly than SmtB. SmtB is known to bind and sense zinc at interhelical four-coordinate, tetrahedral sites across the C-terminal alpha5 helices, while absorption spectroscopy of Co(II)- and Ni(II)-substituted NmtR reveals five- and six-coordinate metal complexes. Site-directed mutagenesis identifies six potential cobalt/ nickel ligands that are obligatory for inducer recognition but not repression by NmtR, four of which (Asp(91), His(93), His(104), His(107)) align with alpha5 ligands of SmtB with two additional His provided by a carboxyl-terminal "extension" (designated alpha5C). Gel retardation assays reveal that zinc does not allosterically regulate NmtR-DNA binding at concentrations where lower affinity cobalt does. These data suggest that two additional ligands form hexacoordinate metal complexes and are crucial for driving allosteric regulation of DNA binding by NmtR, thereby allowing NmtR to preferentially sense metals that favor higher coordination numbers relative to SmtB.