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

摘要 革兰氏阴性菌对β-内酰胺酶的耐药性已成为严重威胁公共卫生的重要因素。 内酰胺类抗生素是世界上使用最广泛和最成功的一类抗生素。这些病原体 主要通过获得水解的β-内酰胺酶蛋白来抵抗多种β-内酰胺类药物 销毁毒品。此外，在药物压力下，β-内酰胺酶正在进化更广泛的β-内酰胺酶 活动了解这些“活性获得”突变的机制对于预测和 遏制其影响。 一个有趣的线索来自临床分离的鲍曼不动杆菌，一种革兰氏阴性病原体 也是全球性的临床灾难鲍曼不动杆菌可将D类β-内酰胺酶OXA-24转化为青霉素 和碳青霉烯类。近年来，临床分离的A.鲍曼不动杆菌的耐药性扩展可追溯到 与底物识别相关的OXA-24柔性片段内的取代突变。这些 结果提出了我们的总体假设，即构象动力学可以影响底物光谱， 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的灵活性-活性研究来调查这一假设， 替代突变体已经建立，在临床上引起“活性获得”表型。我们 研究使用液态NMR来表征游离酶的构象集合， 底物和酰基-酶复合物。 目标1.比较apo OXA-24/40与其临床变体的构象采样。 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 阵 目标3.比较OXA-24/40/配体复合物的构象取样与其 临床变异 对柔性蛋白质区域如何响应抗性扩展突变的预测性理解 仍然是一个公开的挑战。我们提出的研究通过调查以下因素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。我们的研究结果可能会提出新的策略 改进的抑制剂，以及对蛋白质如何进化新功能的新见解。

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.