Metal binding domains in metalloregulatory proteins

描述(由申请人提供)：接触药物和有毒金属会导致获得耐药性机制。细菌的耐药性几乎都是由转录调控的。这项研究的总体目标是深入了解控制细菌耐药性表达的调节蛋白中新的金属结合基序的进化和组织。临床分离的抗性质粒R773和pL258分别携带砷抗性(Ars)和镉抗性(Cad)操纵子，分别编码As(111)/Sb(111)和Pb(11)/Cd(11)/Zn(11)的ATP偶联挤出泵。ArsR和CADC抑制子是两个小的同源金属结合蛋白，分别负责Ars和cad操纵子基因表达的金属调控。在质粒编码的Ars操纵子中，还有第二个不相关的AS(111)/SB(111)反应抑制因子，ARSD。最近的证据表明，ARSD是AS(111)转运型ArsAB泵的金属配位体。具体目的1.金黄色葡萄球菌pL258CADC的结构和功能：在CADC的晶体结构中观察到两种不同类型的金属位点，一种在DNA结合部位，另一种在二聚体界面。将结合分子遗传学、生化、生物物理和结构方法来探索每个位点的性质和功能。 具体目的2.ArsR AS(111)反应抑制物的结构和功能：从结构和功能两个方面进行分析。首先，我们将探讨As(Ill)结合引起的构象变化。其次，将探索As(Ill)结合位点的进化。 具体目标3.ARSD作为金属调节剂和金属配位酮的作用：我们将探索ARSD作为Ars o/p阻滞剂的特性，以及它作为金属配位酮在细胞内将As(Ill)转运到ArsAB As(Ill)挤压泵中的作用。ARS阻遏物和同系物为研究药物和金属耐药性的调控提供了有价值的模型：我们有能力将经典细菌遗传学和现代分子生物学与生化、生物物理和结构方法相结合。

DESCRIPTION (provided by applicant): Exposure to drugs and toxic metals results in the acquisition of resistance mechanisms. Bacterial resistances are nearly all transcriptionally regulated. The overall goal of this study is to gain insights into the evolution and organization of novel metal binding motifs in the regulatory proteins that control expression of bacterial resistances. The clinically isolated resistance plasmids R773 and pl258 carry the arsenical resistance (ars) and cadmium resistance (cad) operons that encode ATP-coupled extrusion pumps for As(lll)/Sb(lll) and Pb(ll)/Cd(ll)/Zn(ll), respectively. The ArsR and CadC repressors are two small homologous metal binding proteins responsible for metalloregulation of gene expression of the ars and cad operons, respectively. In plasmid-encoded ars operons there is a second unrelated As(lll)/Sb(lll)-responsive repressor, ArsD. Recent evidence indicates that ArsD serves as a metallochaperone for the As(lll)- translocating ArsAB pump. Specific Aim 1. Structure and function of the S. aureus plasmid pl258 CadC: Two distinct types of metal sites, one within the DNA binding site and the other at the dimer interface, are observed in the crystal structure of CadC. The properties and function of each site will be explored by using a combination of molecular genetic, biochemical, biophysical and structural approaches. Specific Aim 2. Structure and function of ArsR As(lll)-responsive repressors: Two aspects of ArsR structure and function will be analyzed. First, conformational change induced by As(Ill) binding will be probed. Second, the evolution of As(Ill) binding sites will be explored. Specific Aim 3. Roles of ArsD as a metalloregulator and a metallochaperone: The properties of ArsD that allow it to function as a repressor of the ars o/p will be explored, as will its role as a metallochaperone for intracellular transport of As(Ill) to the ArsAB As(Ill) extrusion pump. The ars repressors and homologues provide valuable models for the study of the regulation of drug and metal resistances: we have the ability to combine classical bacterial genetics and modern molecular biology with biochemical, biophysical and structural approaches.