METAL BINDING DOMAINS IN METALLOREGULATORY PROTEINS

该项目的目标是确定金属结合位点的结构，这些结合位点形成了调节质粒编码细菌抗性的转录抑制因子的诱导剂结合域。与许多药物和抗生素耐药性一样，砷（ars）和镉（cad）抗性操纵子编码转运atp酶，用于挤压As(III)/Sb（III）或Pb(II)/Cd(II)/Zn（II）。ArsR和CadC抑制因子是两个小的同源金属调控蛋白，负责ars和cad操纵子的金属调控基因表达。每个诱导剂结合域所需的残基将被确定。此外，在ars操纵子中还存在第二种As(III)/Sb（III）响应抑制因子ArsD，它与任何已知的金属结合蛋白都没有同源性。ArsR和ArsD形成了一个调节回路，可以感知环境中低浓度和高浓度的类金属。测定As(III)/Sb（III）与ArsD结合的残基。最后，铜（copA）抗性基因编码Cu(I)易位p型atp酶。copA的表达受铜或银的调控。假定的CopR调节蛋白将被鉴定和表征。我们的研究将定义新的金属结合基序。这可能是更普遍的适用性，因为几乎所有与转运相关的耐药性都是转录调控的，而调控蛋白的药物结合基序在很大程度上是未知的。因此，这些外排泵基因的抑制因子为研究传染性细菌抗生素耐药性的调控提供了良好的模型系统。

The goal of this project is determination of the structure of the metal binding sites that form the inducer binding domains of transcriptional repressors that regulate plasmid-encoded bacterial resistances. In common with many drug and antibiotic resistances, the arsenical (ars) and cadmium (cad) resistance operons encode transport ATPases for the extrusion of As(III)/Sb(III) or Pb(II)/Cd(II)/Zn(II). The ArsR and CadC repressors are two small homologous metalloregulatory proteins responsible for metal-regulated gene expression of the ars and cad operons. Residues required for each inducer binding domain will be determined. In addition, in ars operons there is a second As(III)/Sb(III)-responsive repressor, ArsD, which does not exhibit homology to any known metal binding protein. ArsR and ArsD form a regulatory circuit that senses both low and high concentrations of environmental metalloid. The residues involved in As(III)/Sb(III) binding to ArsD will be determined. Finally, the copper (copA) resistance gene encodes a Cu(I)-translocating P-type ATPase. Expression of copA is regulated by copper or silver. The putative CopR regulatory protein will be identified and characterized. Our studies will define new classes of metal binding motifs. This may be more general applicability, since nearly all transport-related drug resistances are transcriptionally regulated, and the drug binding motifs of the regulatory proteins are largely unknown. Thus the repressors of the genes for these efflux pumps provide good model systems for the study of regulation of transmissible bacterial antibiotic resistances.