X-RAY STUDIES ON BACTERIAL MDR REGULATORS

细菌 MDR 调节剂的 X 射线研究

• 批准号: 7188699
• 负责人: RICHARD GERALD BRENNAN
• 金额: $ 7.5万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7188699
• 关键词: Bacillus anthracis; DNA binding protein; Neisseria gonorrhoeae; Staphylococcus aureus; X ray crystallography; antibacterial agents; bacteria infection mechanism; bacterial proteins; calorimetry; circular dichroism; genetic regulation; intermolecular interaction; multidrug resistance; mutant; protein purification; protein structure function; site directed mutagenesis; structural biology; transcription factor

DESCRIPTION (provided by applicant): The emergence of multidrug resistant strains of bacteria is a serious and growing threat to human health. One component of multidrug resistance is the presence of multidrug efflux transporters that expel drugs from the cell thereby keeping their cytosolic levels below toxic concentrations. Nearly all multidrug efflux pump genes are regulated locally by transcription factors that are induced or activated upon binding the very same structurally and chemically dissimilar drugs, which are substrates of the pumps. Thus, these transcription regulators act as intracellular multidrug sensors that increase the number of multidrug efflux pumps when the cell is threatened by an increasing drug dose. Because of their increased solubility, these proteins are outstanding systems to understand the structural mechanisms of multidrug binding and multidrug gene regulation. In this competing renewal application, x-ray crystallographic studies on two multidrug binding transcription factors, QacR from S. aureus and BmrR from B. subtilis and two multidrug efflux pump gene regulators, MtaN from B. subtilis and MtrR from N. gonorrhoeae, will be done to dissect fully the structural mechanisms by which a single protein can bind multiple structurally and chemically dissimilar drugs as well as regulate the expression of multidrug efflux pump genes. The specific aims are: (1) to determine the x-ray structures of QacR and a series of site directed QacR mutants bound to drugs and DNA: (2) to determine the crystal structures of a number of BmrR-drug-DNA complexes and a series of site directed BmrR mutants bound to drugs and DNA as well as the BmrR-bmr operator complex; (3) to determine the structures of site directed mutants of MtaN in order to elucidate its DNA binding mechanism; (4) to crystallize and determine the structures of MtrR bound to its operator site and off DNA. All specific aims will be supplemented with germane DNA and drug binding studies. These data will be used in future structure-based drug design of novel drugs against pathogenic bacteria, including S. aureus, N. gonorrhoeae and potentially B. anthracis.

描述（由申请人提供）：多重耐药细菌菌株的出现对人类健康构成严重且日益严重的威胁。多药耐药性的组成部分之一是存在多药外排转运蛋白，其将药物从细胞中排出，从而将其胞质水平保持在毒性浓度以下。几乎所有多药外排泵基因都受到转录因子的局部调节，这些转录因子在结合结构和化学上完全相同的不同药物（这些药物是泵的底物）时被诱导或激活。因此，这些转录调节剂充当细胞内多药传感器，当细胞受到药物剂量增加的威胁时，它们会增加多药外排泵的数量。由于其溶解度增加，这些蛋白质是了解多药结合和多药基因调控的结构机制的杰出系统。在这一竞争性更新应用中，对两种多药结合转录因子（来自金黄色葡萄球菌的 QacR 和来自枯草芽孢杆菌的 BmrR）以及两种多药外排泵基因调节因子（来自枯草芽孢杆菌的 MtaN 和来自淋病奈瑟菌的 MtrR）的 X 射线晶体学研究将旨在全面剖析单个蛋白质可以结合多种结构和化学上不同的药物以及调节多种药物表达的结构机制外排泵基因。具体目标是：（1）确定 QacR 和一系列与药物和 DNA 结合的定点 QacR 突变体的 X 射线结构：（2）确定许多 BmrR-药物-DNA 复合物的晶体结构和一系列与药物和 DNA 结合的定点 BmrR 突变体以及 BmrR-bmr 操纵子复合物； (3)确定MtaN定点突变体的结构，以阐明其DNA结合机制； (4)结晶并确定与其操纵位点结合并脱离DNA的MtrR的结构。所有具体目标都将辅以密切相关的 DNA 和药物结合研究。这些数据将用于未来针对致病菌（包括金黄色葡萄球菌、淋病奈瑟菌和潜在的炭疽芽孢杆菌）的基于结构的新药设计。