Structure of Beta-lactam resistance regulators

描述（由申请人提供）：金黄色葡萄球菌是最普遍和耐抗生素的致病菌之一。然而，治疗葡萄球菌感染最有效的药物-内酰胺类抗生素，随着葡萄球菌对其耐药性的增加而变得不那么有效。耐药是由一种β -内酰胺酶（由blaZ编码）介导的，该酶可水解青霉素和另一种靶标青霉素结合蛋白（PBP2a，由mecA编码），β -内酰胺类抗生素与青霉素结合蛋白结合较差。当细菌细胞暴露于诱导β -内酰胺时，mecA和blaZ的转录通过MecRl或BlaRl的信号转导被激活。mec （MecRI和Mecl）和bla （BlaRl和Blal）调节因子似乎具有信号转导作用机制，这在原核生物学中是独一无二的，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，而且还可能揭示一个涉及其他分子的诱导级联反应，这些分子可能将p -内酰胺耐药性的调节与基本细胞系统联系起来。本应用程序的主要目标是确定多域积分膜MecRl和/或BlaRl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。这项应用的一个重要的长期目标是为理解抗生素耐药性的分子机制和开发新的治疗方法提供一个结构框架。此外，这些研究还将提供丰富的其他信息，包括膜蛋白的生产、溶解和结晶技术，这些对继续理解整体膜蛋白至关重要。具体目的是：(1)确定完整的多结构域BlaRl/MecRl蛋白的结构；(2)确定合理选择的BlaRl/MecRl蛋白片段的结构；(3)确定BlaRl/MecRl与其同源抑制因子Blal/MecI之间复合物的结构。BlaRl和MecRl的克隆和亚克隆将在大肠杆菌中表达并纯化。结晶实验，以获得衍射质量的晶体使用洗涤剂胶束，三相，囊泡融合，和双胞技术将进行。随后，将使用x射线晶体学确定蛋白质的结构。

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.