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

• 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
• 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

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 革兰氏阴性细菌已成为对公共卫生的严重威胁，因为它们对β-的抵抗力 LACTAM抗生素是全球使用最广泛和成功的抗生素类。这些病原体 主要通过获取β-内酰胺酶蛋白来抵抗多个β-内酰胺 消灭药物。此外，在药物压力下，β-内酰胺酶正在进化的广播β-内酰胺酶 活动。了解这些“活跃性”突变的机制对于预期和 遏制他们的效果。 一个有趣的线索来自baumannii的临床分离株，一种革兰氏阴性病原体 和全球临床冲刷。 Baumannii部署Dβ-内酰胺酶OXA-24，以使青霉素失活 和碳青霉烯。最近，鲍曼尼曲霉的临床分离株可追溯到 与底物识别相关的OXA-24柔性段内的取代突变。这些 结果提出了我们的总体假设，即构象动力学可以影响 D类β-内酰胺酶，特别是在蛋白质表面的柔性识别环中。 因此，我们建议通过OXA-24和 已经建立的替代突变体在诊所引起“活跃性”表型。我们的 投资使用液态NMR来表征自由酶和 WT-24和抗性变体的底物和酰基酶复合物。 AIM 1。将APO OXA-24/40的构象采样与其临床变体的构象采样。 AIM 2。定义由复合物引起的配体构象柔韧性的位点特异性变化 形成。 目标3。比较Oxa-24/40/配体配合物的构象采样 临床变体。 对柔性蛋白质区域如何响应耐药性突变的预测理解 仍然是一个公开挑战。我们拟议的研究通过调查来应对这一挑战 蛋白质灵活性在扩大革兰氏阴性抗生素耐药性方面。我们的结果可能建议新的策略 为了改善抑制剂，以及对蛋白质如何发展新功能的新见解。