Ratiometric pulsed alkylation mass spectrometry as a probe of thiolate reactivity in different metalloderivatives of Staphylococcus aureus pI258 CadC
Ratiometric pulsed alkylation mass spectrometry as a probe of thiolate reactivity in different metalloderivatives of Staphylococcus aureus pI258 CadC
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DOI：
10.1021/bi035668f
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发表时间：
2004-04-06
期刊：
影响因子：
2.9
通讯作者：
Giedroc, DP
中科院分区：
文献类型：
--
作者：
Apuy, JL;Busenlehner, LS;Giedroc, DP
金属传感金属调节配合物中金属配体的配位结构和反应性可能很好地决定了它们的生物学性质。在这里，我们使用比率脉冲烷基化质谱（rPA-MS）的技术来探测由金黄色葡萄球菌质粒pI 258编码的CadC，金属调节的cad操纵子的转录抑制子的不同金属衍生物形成的金属配位络合物的结构和反应性。cad操纵子提供对大的亲硫重金属污染物（包括Cd（II）、Pb（II）和Bi（III））的抗性。两个半胱氨酸，一个不变的Cys 7和一个保守的Cys 11，由三个氨基酸分开的N-末端附近的每个亚基内的二聚体CadC，捐赠两个四个配位键镉（II）和铋（III），相反，Cys 11，但不是Cys 7，被排除在三角形的铅（II）复合物。rPA-MS显示，Cys 7在所有金属络合物中都受到强烈的烷基化保护，Pb（II）是最有效的，相对于apo-CadC，k（C7）（app）降低了约1000倍;相反，Cys 11的反应性与apo-CadC的反应性没有区别，与S-3配位络合物一致。只有在由Cd（II）和Bi（III）形成的四硫醇盐络合物中，Cys 11的反应性才明显降低，但仅降低大于或等于10倍。这些数据表明，Cys 11-S-金属配位键或在三方Pb（II）络合物中的配位螯合物的一侧定义了螯合物中的“弱点”，因此可能为潜在的金属配体交换反应提供一个进入位点，该反应对体内金属抗性很重要。相反，Cys 7与所有诱导金属形成紧密的配位键，这与其在操纵子/启动子结合的金属调节中作为关键变构配体的作用一致。
The coordination structure and reactivities of metal ligands in metal-sensing metalloregulatory coordination complexes may well dictate their biological properties. Here, we use the technique of ratiometric pulsed alkylation mass spectrometry (rPA-MS) to probe the structure and reactivities of metal coordination complexes formed by different metalloderivatives of Staphylococcus aureas plasmid pI258-encoded CadC, the metal-regulated transcriptional repressor of the cad operon. The cad operon provides resistance to large thiophilic heavy metal pollutants including Cd(II), Pb(II), and Bi(III). Two cysteines, an invariant Cys7 and a conserved Cys11, separated by three amino acids near the N-terminus of each subunit within dimeric CadC, donate two of the four coordination bonds to Cd(II) and Bi(III); in contrast, Cys11, but not Cys7, is excluded from the trigonal Pb(II) complex. rPA-MS reveals that Cys7 is strongly protected from alkylation in all metal complexes, Pb(II) being most effective, reducing k(C7)(app) by similar to 1000- fold relative to apo-CadC; in contrast, the reactivity of Cys11 is indistinguishable from that of apo-CadC, consistent with an S-3 coordination complex. Only in the tetrathiolate complexes formed by Cd(II) and Bi(III) is the reactivity of Cys11 appreciably reduced, but only by greater than or equal to 10-fold. These data suggest that the Cys11-S--metal coordination bond or that side of the coordination chelate in the trigonal Pb(II) complex defines a "weak point" in the chelate and thus might provide an entry site for potential metal ligand exchange reactions important for metal resistance in vivo. In contrast, Cys7 forms a tight coordination bond with all inducing metals, consistent with its role as a critical allosteric ligand in the metalloregulation of the operator/promoter binding.