Structure of Beta-lactam resistance regulators

描述(申请人提供)：金黄色葡萄球菌是最常见和对抗生素耐药的病原菌之一。然而，作为治疗葡萄球菌感染最有效的治疗方法，β-内酰胺类抗生素随着葡萄球菌对它们的耐药性增加而变得不那么有效。耐药性是由一种β-内酰胺酶(由Blaz编码)和另一种靶点青霉素结合蛋白(PBP2a，由mecA编码)介导的，而β-内酰胺类抗生素很难与其结合。当细菌细胞暴露于诱导性的β-内酰胺时，mecA和Blaz的转录通过MecRl或BlaR1的信号转导被激活。Mec(Mecri和Mecl)和bla(BlaRl和Blal)调节子似乎具有在原核生物中独一无二的信号转导作用机制，但其作用机制尚不清楚，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，还可能揭示一个涉及其他分子的诱导级联反应，这些分子可能会将β-内酰胺耐药的调节与必要的细胞系统联系起来。这一应用的主要目标是确定多结构域整合膜MecRl和/或BlaRl蛋白的晶体结构，从而为了解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。这一应用的一个重要的长期目标是为理解抗生素耐药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、增溶和结晶技术，这些技术对于继续了解完整的膜蛋白至关重要。其具体目的是：(1)确定完整的多结构域BlaRl/MecRl蛋白的结构；(2)合理选择BlaRl/MecR1蛋白片段的结构；(3)确定BlaRl/MecRl与其同源阻滞剂Blal/MecI之间的复合体的结构。BlaR1和MecR1的克隆和亚克隆将在大肠杆菌中表达和纯化。将进行结晶实验，使用洗涤剂胶束技术、立方相技术、囊泡融合技术和双孔技术获得衍射级晶体。随后，将使用X射线结晶学来确定蛋白质的结构。

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.