Structure of Beta-lactam resistance regulators

β-内酰胺耐药调节剂的结构

• 批准号: 7229829
• 负责人: Martin K Safo
• 金额: $ 21.7万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7229829
• 关键词: Adopted; Antibiotic Resistance; Antibiotics; Bacillus licheniformis; Bacteriology; Binding; Biological; Biology; Cells; Chimera organism; Complex; Crystallization; Cytoplasmic Structures; Cytoplasmic Tail; DNA; Data Analyses; Detergents; Endopeptidases; Escherichia coli; Exhibits; Genetic Transcription; Genus staphylococcus; Goals; Hydrolysis; Integral Membrane Protein; Knowledge; Lactamase; Lactams; Link; Mediating; Membrane; Membrane Proteins; Micelles; Molecular; Molecular Conformation; Monobactams; Penicillin-Binding Proteins; Penicillins; Peptide Hydrolases; Phase; Production; Protease Domain; Proteins; Publishing; Regulation; Research Personnel; Resistance; Resistance development; Signal Transduction; Signaling Molecule; Solutions; Specificity; Staphylococcal Infections; Staphylococcus aureus; Structure; System; Techniques; Transducers; Transmembrane Domain; Vesicle; Water; X-Ray Crystallography; beta-Lactam Resistance; beta-Lactamase; beta-Lactams; in vivo; insight; interest; novel therapeutics; pathogenic bacteria; programs; protein structure; research study; sensor

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（由申请方提供）：金黄色葡萄球菌是最普遍和最耐药的病原菌之一。然而，β-内酰胺类抗生素是治疗葡萄球菌感染的最有效疗法，随着葡萄球菌对它们的耐药性增加，其效果越来越差。耐药性由β-内酰胺酶（由blaZ编码）介导，该酶水解青霉素和替代靶点青霉素结合蛋白（PBP 2a，由mecA编码），β-内酰胺抗生素与其结合较差。当细菌细胞暴露于诱导性β-内酰胺时，mecA和blaZ的转录通过经由MecRl或BlaRl的信号转导而被激活。mec（MecRI和Mecl）和bla（BlaRl和Blal）调节剂似乎具有在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，但也可能揭示涉及其他分子的诱导级联反应，可以连接调节β-内酰胺抗性的基本细胞系统。本申请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，这将提供对负责信号接收和信号转导的分子部分之间的关系的独特见解。本申请的一个重要的长期目标是提供一个结构框架，用于理解抗生素耐药性和新疗法开发中涉及的分子机制。此外，这些研究还将提供丰富的其他信息，包括膜蛋白生产，溶解和结晶技术，这对继续了解膜蛋白是至关重要的。具体目标是：（1）完整的多结构域BlaR 1/MecR 1蛋白的结构测定，（2）合理选择的BlaR 1/MecR 1蛋白片段的结构测定，和（3）BlaR 1/MecR 1和它们的同源阻遏物Bla 1/MecI之间的复合物的结构测定。BlaR 1和MecR 1的克隆和亚克隆将在E. coli中进行纯化。结晶实验，以获得衍射质量的晶体，使用洗涤剂胶束，反相，囊泡融合，和bicelle技术将进行。随后，将使用X射线晶体学确定蛋白质的结构。