X-RAY STUDIES ON BACTERIAL MDR REGULATORS

描述：(逐字摘自申请人的摘要)出现 细菌多药耐药(MDR)严重威胁人类健康。 多药耐药的一个关键因素是膜结合的转运蛋白 多种、化学成分多样的药物来自细菌细胞。结构性的 这些蛋白质识别不同药物的机制完全是 未知，主要是因为它们是完整的膜蛋白，因此 很难提纯。细菌也有第二类多药结合。 对他们的多药耐药表型至关重要的蛋白质。这些 胞浆蛋白是多药转运蛋白的转录调节因子 基因。枯草芽孢杆菌的一种调节因子是BmrR。BmrR戏剧性 增加多药转运蛋白基因BMR的转录，只有在 结合药物是BMR底物，但已侵入细胞质。因此，BmrR 起到第二道防线的作用，防止药物进入细胞 目标。BmrR-药物和BmrR-DNA+药物络合物的结构也将揭示 Merr家族成员的转录调控机制，类到 BmrR属于哪一个。第二个多药结合调节蛋白是QacR 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，这也是 QacA转运蛋白，诱导QacR，并降低qacA基因的表达，从而为 细菌具有更多的转运体，以抵御潜在的致命药物剂量。 结构研究将揭示多药物结合和 QacR的转录抑制机制。有趣的是，QacR和BmrR 显示其重叠的药物结合特性和结构 同种药物复合体将揭示它们之间的异同 多药结合机制。这项拨款提案有四个具体目标。至 结晶并确定C-末端的结构，多药结合 BmrR的结构域，也就是所谓的BRC，与许多显示出 广泛的结合亲和力。BRC提供了高分辨率的优势， 这将极大地帮助分析BrnrR的药物结合机制。至 BmrR药物DNA和BmrR-DNA的结晶及X射线结构测定 复合体。枯草杆菌的结晶和X射线结构的测定 全球MD调节剂、mTan及其DNA复合体。使之具体化并确定 QacR-药物和QacR-DNA络合物的X射线结构。的宏伟目标 这项工作是为了提供一个完整的机制的了解 BrnrR、mTan和QacR与多药结合及其对MerR和QacR的基因调控 TETR/CAMR家族成员。这些数据将是未来基于结构的 抗致病菌新药的药物设计。

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.