Metal binding domains in metalloregulatory proteins

描述（由申请人提供）：暴露于药物和有毒金属会导致获得耐药性机制。细菌耐药性几乎都是受转录调控的。本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型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(ll)/Sb（ll）和Pb(ll)/Cd(ll)/Zn（ll）的atp偶联挤出泵。ArsR和CadC抑制因子是两个小的同源金属结合蛋白，分别负责ars和cad操纵子的金属调控基因表达。在质粒编码的ars操纵子中，存在第二个不相关的As(ll)/Sb（ll）应答抑制子ArsD。最近的证据表明，ArsD作为as (ll)-转运ArsAB泵的金属伴侣。具体目标金黄色葡萄球菌质粒pl258 CadC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结合位点内，另一种在二聚体界面处。每个位点的性质和功能将通过使用分子遗传学，生物化学，生物物理和结构方法的组合来探索。

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.