Busenlehner Laura S, Giedroc David P
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
J Inorg Biochem. 2006 May;100(5-6):1024-34. doi: 10.1016/j.jinorgbio.2006.01.002. Epub 2006 Feb 17.
The mechanisms by which metal ions are sensed in bacterial cells by metal-responsive transcriptional regulators (metal sensor proteins) may be strongly influenced by the kinetics of association and dissociation of specific metal ions with specific metalloregulatory targets. Staphylococcus aureus pI258-encoded CadC senses toxic metal pollutants such as Cd(II), Pb(II) and Bi(III) with very high thermodynamic affinities ( approximately 10(12)M(-1)) in forming either distorted tetrahedral (Cd/Bi) or trigonal (Pb) coordination complexes with cysteine thiolate ligands derived from the N-terminal domain (Cys7/11) and a pair of Cys in the alpha4 helix (Cys58/60). We show here that metal ion binding to this site (denoted the alpha3N or type 1 metal site) is characterized by two distinct kinetic phases, a fast bimolecular encounter phase and a slower intramolecular conformational transition. Metal association rates are fast ( approximately 10(5)-10(7)M(-1)s(-1)) and strongly dependent on the metal ion type in a manner that correlates with metal specificity in vivo. In contrast, the observed rate of the slower isomerization step is independent of the metal ion type (2.8+/-0.4s(-1)) but is reduced 6-fold upon substitution of Cys7, a key metal ligand that drives allosteric negative regulation of DNA binding. Chelator (EDTA)-mediated metal dissociation rates from the alpha3N site are extremely slow (10(-4)s(-1)). Where observable dissociation can be observed, a ternary CadC-metal ion-chelator complex is invoked, suggesting that metal-ligand exchange may be an important factor in metal sensing and resistance in the cell.
金属响应转录调节因子（金属传感器蛋白）在细菌细胞中感知金属离子的机制，可能会受到特定金属离子与特定金属调节靶点结合和解离动力学的强烈影响。金黄色葡萄球菌pI258编码的CadC以非常高的热力学亲和力（约10¹²M⁻¹）感知有毒金属污染物，如Cd(II)、Pb(II)和Bi(III)，形成扭曲的四面体（Cd/Bi）或三角（Pb）配位络合物，其配体为来自N端结构域的半胱氨酸硫醇盐配体（Cys7/11）和α4螺旋中的一对半胱氨酸（Cys58/60）。我们在此表明，金属离子与该位点（称为α3N或1型金属位点）的结合具有两个不同的动力学阶段，一个快速的双分子相遇阶段和一个较慢的分子内构象转变阶段。金属结合速率很快（约为10⁵ - 10⁷M⁻¹s⁻¹），并且在很大程度上取决于金属离子类型，这种方式与体内的金属特异性相关。相比之下，观察到的较慢异构化步骤的速率与金属离子类型无关（2.8±0.4s⁻¹），但在取代驱动DNA结合变构负调控的关键金属配体Cys7后，速率降低了6倍。螯合剂（EDTA）介导的金属离子从α3N位点的解离速率极慢（10⁻⁴s⁻¹）。在可以观察到解离的情况下，会形成三元CadC - 金属离子 - 螯合剂复合物，这表明金属 - 配体交换可能是细胞中金属感知和抗性的一个重要因素。
