Cavet Jennifer S, Graham Alison I, Meng Wenmao, Robinson Nigel J
Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle NE2 4HH, United Kingdom.
J Biol Chem. 2003 Nov 7;278(45):44560-6. doi: 10.1074/jbc.M307877200. Epub 2003 Aug 25.
We report a cadmium- and lead-detecting transcriptional repressor from Mycobacterium tuberculosis designated CmtR. Two genes were co-transcribed with cmtR, one encoding a deduced P1 type ATPase. Purified CmtR bound to the cmt operator-promoter, and repression of transcription was lost after introduction of a stop codon into cmtR. Assays of metal-dependent expression from cmt and nmt operator-promoters established that the metal specificity of CmtR in vivo was perfectly inverted relative to the nickel-cobalt sensor NmtR from the same organism, with CmtR totally insensitive to Co(II) or Ni(II) and NmtR totally insensitive to Cd(II) or Pb(II). Absorption spectroscopy of Cd(II)-, Co(II)-, and Ni(II)-substituted CmtR revealed S- to metal-charge-transfer which was absent in NmtR, providing diagnostic metal-difference spectra that discriminated between metal-binding to these two proteins. Ni(II)-binding isothermal titrations of CmtR are complex, with Kapp = 1.8 x 10(4) m(-1) for site1, three orders of magnitude weaker than KNi for NmtR. Mixing equimolar apo-NmtR and apo-CmtR with 0.9 equivalents of Cd(II) gave Cd(II)-dependent difference spectra almost identical to Cd(II)0.9-CmtR. Thus, Cd(II) bound to CmtR in preference to NmtR, whereas the converse was true for Ni(II); this correlates faithfully with and provides a simplistic basis for metal-sensing preferences. In contrast, CmtR and NmtR had similar affinities for Co(II), and alternative explanations for Co(II) sensitivities are invoked. ArsR-SmtB repressors detect metals through derivatives of one or both of two possible allosteric sites at either carboxyl-terminal alpha5 helices or helix alpha3 proximal to the DNA-binding site. Unexpectedly, neither site was required for inducer recognition by CmtR. The mutants in potential metal ligands in, or near, these regions, Cys4, Cys35, Asp79, His81, Asp97, Asp99, Glu105, Glu111, and Glu114, retained both repression and inducer recognition. Crucially, substitution of Cys57, Cys61, and Cys102 with Ser revealed that each of these three residues is obligatory for Cd(II) detection, and this defines completely new sensory sites.
我们报道了一种来自结核分枝杆菌的镉和铅检测转录抑制因子，命名为CmtR。有两个基因与cmtR共转录，其中一个编码一种推测的P1型ATP酶。纯化的CmtR与cmt操纵子 - 启动子结合，在cmtR中引入终止密码子后转录抑制作用丧失。对cmt和nmt操纵子 - 启动子的金属依赖性表达分析表明，CmtR在体内的金属特异性相对于来自同一生物体的镍 - 钴传感器NmtR完全相反，CmtR对Co(II)或Ni(II)完全不敏感，而NmtR对Cd(II)或Pb(II)完全不敏感。对Cd(II)、Co(II)和Ni(II)取代的CmtR进行吸收光谱分析，发现存在S到金属的电荷转移，而NmtR中不存在这种情况，这提供了可区分这两种蛋白质与金属结合的诊断性金属差异光谱。CmtR与Ni(II)结合的等温滴定量复杂，位点1的Kapp = 1.8×10⁴ m⁻¹，比NmtR的KNi弱三个数量级。将等摩尔的脱辅基NmtR和脱辅基CmtR与0.9当量的Cd(II)混合，得到的Cd(II)依赖性差异光谱几乎与Cd(II)₀.₉ - CmtR相同。因此，Cd(II)优先与CmtR结合而非NmtR，而对于Ni(II)则相反；这与金属传感偏好忠实地相关并提供了一个简单的基础。相比之下，CmtR和NmtR对Co(II)具有相似的亲和力，并对Co(II)敏感性提出了其他解释。ArsR - SmtB抑制因子通过羧基末端α5螺旋或靠近DNA结合位点的α3螺旋这两个可能的变构位点中一个或两个的衍生物来检测金属。出乎意料的是，CmtR识别诱导剂不需要这两个位点中的任何一个。这些区域内或附近潜在金属配体中的突变体，即Cys4、Cys35、Asp79、His81、Asp97、Asp99、Glu105、Glu111和Glu114，保留了抑制作用和诱导剂识别能力。至关重要的是，将Cys57、Cys61和Cys102替换为Ser表明，这三个残基中的每一个对于检测Cd(II)都是必需的，这定义了全新的传感位点。
