Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA
Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA
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DOI：
10.1016/j.jmb.2005.12.019
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发表时间：
2006-03-10
影响因子：
5.6
通讯作者：
Giedroc, DP
中科院分区：
文献类型：
--
作者：
Pennella, MA;Arunkumar, AI;Giedroc, DP
最近对金属调节蛋白的研究表明，宿主细胞质中的配位数/几何构型和金属离子的有效性是决定生物特异性的关键因素。在这里，我们研究了金黄色葡萄球菌CHARAα5敏感位点的单个金属配体(Asp84、His86、His97‘和His100’)对体外金属离子结合亲和配位构型的贡献，以及变构对DNA操纵子/启动子区域结合的负调控。当~gt；=10(3)M-1时，所有配体取代突变体的金属离子结合亲和力(K-Me)均显著降低。Asp84和His97的取代在金属结合时产生非天然配位几何构型，并且在金属与DNA结合的变构偶联(Delta G(偶联)接近0kcal mol(-1))中不起作用。相比之下，His86和His100可以很容易地被潜在的配位(Asp，Glu)和较差的配位(Asn，Gln)残基取代，这些突变体中保留了显著的天然类四面体金属配位构型，导致了强功能偶联(Delta G(偶联)&Gt；=+3.0kcal mol(-1))。野生型和突变型CHARRA的H-1-N-15异核单量子相干(HSQC)谱表明，所有H86和H100替换突变体在结合锌(II)时都经历了4度结构转换，而D84N、H97N和H97D CHARRA则没有。因此，只有那些在金属结合时保留野生型JARA的某些四面体配位几何特征的变异JARA才能够驱动与体外DNA结合的变构调节有关的4度结构构象变化，而与K-Me的大小无关。(C)2006爱思唯尔有限公司。保留所有权利。
Recent studies on metalloregulatory proteins suggest that coordination number/geometry and metal ion availability in a host cytosol are key determinants for biological specificity. Here, we investigate the contribution that individual metal ligands of the alpha 5 sensing site of Staphylococcus aureus CzrA (Asp84, His86, His97', and His100') make to in vitro metal ion binding affinity coordination geometry, and allosteric negative regulation of DNA operator/promoter region binding. All ligand substitution mutants exhibit significantly reduced metal ion binding affinity (K-Me) by >= 10(3) M-1. Substitutions of Asp84 and His97 give rise to non-native coordination geometries upon metal binding and are non-functional in allosteric coupling of metal and DNA binding (Delta G(coupling) approximate to 0 kcal mol(-1) In contrast, His86 and His100 could be readily substituted with potentially liganding (Asp, Glu) and poorly liganding (Asn, Gln) residues with significant native-like tetrahedral metal coordination geometry retained in these mutants, leading to strong functional coupling (Delta G(coupling) >= + 3.0 kcal mol(-1)). H-1-N-15 heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4 degrees structural switching on binding Zn(II), while D84N, H97N and H97D CzrAs do not. Thus, only those variant CzrAs that retain some tetrahedral coordination geometry characteristic of wild-type CzrA upon metal binding are capable of driving 4 degrees structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of K-Me. (c) 2006 Elsevier Ltd. All rights reserved.