Defining flexibility and activity relationships for gram-negative antibiotic resistance proteins

定义革兰氏阴性抗生素抗性蛋白的灵活性和活性关系

基本信息

批准号：
9898388
负责人：
JEFFREY W PENG
金额：
$ 27.04万
依托单位：
UNIVERSITY OF NOTRE DAME
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9898388
关键词：
Acinetobacter baumannii Address Amino Acid Substitution Antibiotic Resistance Antibiotics Behavior Binding Carbapenems Clinic Clinical Complex Evolution Goals Gram-Negative Bacteria Hydroxyl Radical Investigation Knowledge Ligands Liquid substance Membrane Proteins Modeling Molecular Conformation Monobactams Multienzyme Complexes Mutation Nosocomial Infections Parents Pathogenicity Penicillins Pharmaceutical Preparations Phenotype Positioning Attribute Principal Investigator Process Protein Region Proteins Public Health Research Resistance Roentgen Rays Role Sampling Scourge Site Source Structure Substrate Interaction Surface Variant beta-Lactam Resistance beta-Lactamase beta-Lactams carbapenem resistance cohort deacylation enzyme substrate flexibility improved inhibitor/antagonist insight mutant pathogen pathogenic bacteria pressure programs protein function receptor response

项目摘要

ABSTRACT Gram-negative bacteria have become a serious threat to public health because their resistance to β- lactam antibiotics, the most widely used and successful class of antibiotics worldwide. These pathogens resist multiple β−lactams chiefly through the acquisition of β−lactamase proteins, which hydrolytically destroy the drug. Moreover, under drug pressure, the β−lactamases are evolving broader β-lactamase activity. Understanding the mechanisms for these “gain-of-activity” mutations is crucial for anticipating and curbing their effects. An intriguing clue has come from clinical isolates of Acinetobacter baumannii, a Gram-negative pathogen and a global clinical scourge. A baumannii deploys a Class D β-lactamase, OXA-24, to inactivate penicillins and carbapenems. Recently, clinical isolates of A. baumannii with expanded resistance were traced to substitution mutations within flexible segments of OXA-24 associated with substrate recognition. These results raise our overall hypothesis that conformational dynamics can influence the substrate spectrum of Class-D β-lactamases, specifically, in the flexible recognition loops at the protein surface. We therefore propose investigating this hypothesis through flexibility-activity studies of OXA-24 and substitution mutants already established to cause “gain-of-activity” phenotypes in the clinic. Our investigations use liquid state NMR to characterize the conformational ensembles of the free enzyme and substrate, and acyl-enzyme complex, for WT-OXA-24 and resistant variants. Aim 1. Compare the conformational sampling of apo OXA-24/40 with that of its clinical variants. Aim 2. Define the site-specific changes in ligand conformational flexibility caused by complex formation. Aim 3. Compare the conformational sampling of the OXA-24/40/ligand complexes with those of its clinical variants. A predictive understanding of how flexible protein regions respond to resistance-expanding mutations remains an open challenge. Our proposed research answers this challenge via investigations into the role of protein flexibility in expanding gram-negative antibiotic resistance. Our results may suggest new strategies for improved inhibitors, and new insights into how proteins evolve new functions.

抽象的革兰氏阴性菌因其对 β- 的抵抗力而成为对公众健康的严重威胁。内酰胺抗生素是全世界使用最广泛、最成功的一类抗生素。主要通过获得β-内酰胺酶蛋白来抵抗多种β-内酰胺，β-内酰胺酶蛋白会水解此外，在药物压力下，β-内酰胺酶正在进化出更广泛的β-内酰胺酶。了解这些“活性获得”突变的机制对于预测和预测至关重要。遏制它们的影响。一个有趣的线索来自革兰氏阴性病原体鲍曼不动杆菌的临床分离株鲍曼不动杆菌利用 D 类 β-内酰胺酶 OXA-24 来灭活青霉素。最近，耐药性扩大的鲍曼不动杆菌的临床分离株被追踪到。 OXA-24 柔性片段内与底物识别相关的替代突变。结果提出了我们的总体假设，即构象动力学可以影响底物谱 D 类 β-内酰胺酶，特别是在蛋白质表面的灵活识别环中。因此，我们建议通过 OXA-24 和 OXA-24 的灵活性活性研究来调查这一假设。我们已经在临床上建立了可引起“活性增强”表型的替代突变体。研究使用液态核磁共振来表征游离酶的构象集合 WT-OXA-24 和抗性变体的底物和酰基酶复合物。目标 1. 将 apo OXA-24/40 的构象采样与其临床变体的构象采样进行比较。目标 2. 定义由复合物引起的配体构象灵活性的位点特异性变化形成。目标 3. 将 OXA-24/40/配体复合物的构象采样与它的构象采样进行比较临床变异。对柔性蛋白质区域如何响应耐药性扩大突变的预测性理解我们提出的研究通过调查的作用来回答这一挑战。蛋白质在扩大革兰氏阴性抗生素耐药性方面的灵活性我们的结果可能会提出新的策略。改进抑制剂，以及对蛋白质如何进化新功能的新见解。