X-RAY STUDIES ON BACTERIAL MDR REGULATORS

细菌 MDR 调节剂的 X 射线研究

• 批准号: 6226262
• 负责人: RICHARD GERALD BRENNAN
• 金额: $ 28.93万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 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 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6226262
• 关键词: Bacillus subtilis; Staphylococcus aureus; X ray crystallography; antibacterial agents; bacteria infection mechanism; bacterial proteins; chemical binding; crystallization; gene expression; genetic regulation; intermolecular interaction; membrane transport proteins; multidrug resistance; nucleic acid structure; protein structure function; stereochemistry; structural biology; transcription factor

DESCRIPTION: (Verbatim from the Applicant's Abstract) The emergence of bacterial multidrug resistance (MDR) poses a serious threat to human health. One key factor underlying MDR is membrane bound transporters that extrude multiple, chemical diverse drugs from the bacterial cell. The structural mechanism by which these proteins recognize dissimilar drugs is completely unknown, primarily because they are integral membrane proteins and thus more difficult to purify. Bacteria also have a second class of multidrug binding proteins that is central to their multidrug resistant phenotypes. These cytosolic proteins are transcription regulators of the multidrug transporter genes. One regulator from Bacillus subtilis is BmrR. BmrR dramatically increases transcription of the multidrug transporter gene, bmr, only after binding drugs that are Bmr substrates but have invaded the cytosol. Thus, BmrR acts as a second line of defence against drugs from reaching their cellular targets. Structures of BmrR-Drug and BmrR-DNA+Drug complexes will also reveal the transcription regulation mechanism of the MerR family member, the class to which BmrR belongs. A second multidrug binding regulatory protein is QacR from Staphylococcus aureus. QacR represses the qacA and multidrug transporter gene and belongs to the TetR/CamR family. Drugs, which are also substrates of the QacA transporter, induce QacR and derepress the qacA gene thereby providing the bacterium with the more transporters to fend off potentially lethal drug doses. Structural studies will unveil the underpinnings of the multidrug binding and transcription repression mechanisms of QacR. Interestingly, QacR and BmrR display overlapping drug binding specificities and structures of their same-drug complexes will reveal the similarities and differences of their multidrug binding mechanisms. This grant proposal has four specific aims. To crystallize and determine the structures of the C-terminal, multidrug binding domain of BmrR, the so named BRC, bound to a number of drugs that display a wide range of binding affinities. BRC offers the advantages of high resolution, which will greatly aid the analysis of the drug binding mechanism of BrnrR. To crystallize and determine the x-ray structures of BmrRDrug-DNA and BmrR-DNA complexes. To crystallize and determine the x-ray structures of the B. subtilis global MD regulator, MtaN and its DNA complexes. To crystallize and determine the x-ray structures of QacR-drug and QacR-DNA complexes. The broad goals of this work are to provide a complete understanding of the mechanisms of multidrug binding by BrnrR, MtaN and QacR and gene regulation of these MerR and TetR/CamR family members. These data will be key to the future structure-based drug design of novel drugs against pathogenic bacteria.

描述：（逐字来自申请人的摘要） 细菌的多药耐药（MDR）严重威胁着人类的健康。 MDR的一个关键因素是膜结合转运蛋白， 从细菌细胞中提取多种化学药物。结构性 这些蛋白质识别不同药物的机制完全是 未知，主要是因为它们是膜蛋白，因此更多 难以净化。细菌也有第二类多药结合 这些蛋白质是其多药耐药表型的核心。这些 胞浆蛋白是多药转运蛋白的转录调节因子 基因.来自枯草芽孢杆菌的一种调节剂是BmrR。BmrR显著 增加多药转运蛋白基因BMR的转录，只有在 结合作为Bmr底物但已侵入胞质溶胶的药物。因此，BmrR 作为第二道防线，阻止药物进入细胞， 目标的BmrR-药物和BmrR-DNA+药物复合物的结构也将揭示 MerR家族成员的转录调控机制， BmrR属于哪一种。第二种多药物结合调节蛋白是QacR，来自 金黄色葡萄球菌。QacR抑制qacA和多药转运蛋白基因 属于TetR/CamR家族。药物，也是药物的底物， QacA转运蛋白，诱导QacR和去抑制qacA基因，从而提供 细菌有更多的转运蛋白来抵御潜在的致命药物剂量。 结构研究将揭示多药结合的基础， QacR的转录抑制机制。有趣的是，QacR和BmrR 显示重叠的药物结合特异性和它们的结构 相同的药物复合物将揭示其相似性和差异性， 多药结合机制这项拨款建议有四个具体目标。到 结晶并确定C-末端的结构， BmrR的结构域，即所谓的BRC，与许多药物结合， 广泛的结合亲和力。BRC提供了高分辨率的优点， 这将大大有助于分析BrnrR的药物结合机制。到 结晶并确定BmrRDrug-DNA和BmrR-DNA的X射线结构 配合物结晶并确定B的X射线结构。枯草 全球MD调节因子MtaN及其DNA复合物。结晶并确定 QacR-药物和QacR-DNA复合物的X射线结构。的广泛目标 这项工作是为了提供一个完整的了解机制， BrnrR、MtaN和QacR的多药结合以及这些MerR的基因调控， TetR/CamR家族成员。这些数据将是未来基于结构的 抗病原菌新药的药物设计。