Structure of Beta-lactam resistance regulators

β-内酰胺耐药调节剂的结构

• 批准号: 7014710
• 负责人: Martin K Safo
• 金额: $ 18.63万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014710
• 关键词: Escherichia coli; Staphylococcus aureus; X ray crystallography; bacterial proteins; beta lactam antibiotic; beta lactamase; biological signal transduction; drug resistance; hydrolysis; protein purification; protein structure function

DESCRIPTION (provided by applicant): Staphylococcus aureus is among the most prevalent and antibiotic-resistant of pathogenic bacteria. However, beta-lactam antibiotics, the most effective therapy for treating staphylococcal infections, are becoming less effective as resistance to them among staphylococci increases. Resistance is mediated by a beta-lactamase (encoded by blaZ) that hydrolyzes penicillins and an alternate target, penicillin binding protein (PBP2a, encoded by mecA), to which beta-lactam antibiotics bind poorly. Transcription of mecA and blaZ is activated by signal transduction through either MecRl or BlaRl when the bacterial cell is exposed to an inducing beta-lactam. The mec (MecRI and Mecl) and bla (BlaRl and Blal) regulators seem to have a signal transduction mechanism of action that is unique in prokaryotic biology but its mechanism is unclear, due in part to lack of structural information. Answers to this question not only solve an interesting biological question but may also uncover an induction cascade involving other molecules that could link regulation of P-lactam resistance to essential cellular systems. The major goal of this application is to determine the crystal structures of the multidomain integral membrane MecRl and/or BlaRl proteins that will provide a unique insight into the relationship between the parts of the molecules that are responsible for signal reception and signal transduction. An important long-term objective of this application is to provide a structural framework for understanding the molecular mechanisms involved in antibiotic resistance and the development of novel therapeutics. Moreover, the studies will also provide a wealth of other information, including membrane protein production, solubilization and crystallization techniques that are crucial to the continued understanding of integral membrane proteins. The specific aims are: (1) Structure determination of the intact multidomain BlaRl/MecRl proteins, (2) structure determination of rationally chosen fragments of the BlaRl/MecRl protein, and (3) structure determination of complex between BlaRl/MecRl and their homologous repressers Blal/MecI. The clones and subclones of BlaRl and MecRl will be expressed in E. coli and purified. Crystallization experiments to obtain diffraction-quality crystals using the detergent-micelle, cubic-phase, vesicle-fusion, and bicelle techniques will be undertaken. Subsequently, the structures of the proteins will be determined using X-ray crystallography.

描述（由申请人提供）：金黄色葡萄球菌是最常见且对抗生素具有耐药性的病原菌之一。然而，β-内酰胺抗生素是治疗葡萄球菌感染最有效的疗法，但随着葡萄球菌对其耐药性的增加，其效果正变得越来越差。耐药性是由水解青霉素的 β-内酰胺酶（由 blaZ 编码）和替代靶点青霉素结合蛋白（PBP2a，由 mecA 编码）介导的，β-内酰胺抗生素与该蛋白的结合效果较差。当细菌细胞暴露于诱导性β-内酰胺时，通过MecR1或BlaR1的信号转导激活mecA和blaZ的转录。 mec（MecRI 和 Mecl）和 bla（BlaRl 和 Bal）调节因子似乎具有原核生物学中独特的信号转导作用机制，但其机制尚不清楚，部分原因是缺乏结构信息。这个问题的答案不仅解决了一个有趣的生物学问题，而且还可能揭示涉及其他分子的诱导级联反应，这些分子可能将β-内酰胺抗性的调节与重要的细胞系统联系起来。本申请的主要目标是确定多域整合膜MecR1和/或BlaR1蛋白的晶体结构，这将为负责信号接收和信号转导的分子部分之间的关​​系提供独特的见解。该应用的一个重要的长期目标是提供一个结构框架，以了解抗生素耐药性和新疗法开发所涉及的分子机制。此外，这些研究还将提供大量其他信息，包括膜蛋白生产、溶解和结晶技术，这些对于持续了解完整膜蛋白至关重要。具体目标是：(1)完整多结构域BlaRl/MecRl蛋白的结构测定，(2)BlaRl/MecRl蛋白的合理选择片段的结构测定，以及(3)BlaRl/MecRl与其同源阻遏物Blal/MecI之间的复合物的结构测定。 BlaR1和MecR1的克隆和亚克隆将在大肠杆菌中表达并纯化。将进行结晶实验，以使用去污剂胶束、立方相、囊泡融合和双束技术获得衍射质量的晶体。随后，将使用 X 射线晶体学确定蛋白质的结构。