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

摘要 革兰氏阴性细菌已经成为对公共健康的严重威胁，因为他们对β的耐药性- 内酰胺类抗生素，全球使用最广泛、最成功的一类抗生素。这些病原体 对多重β−内酰胺类抗生素的抵抗主要是通过获取β−内酰胺酶蛋白来实现的，该酶蛋白可水解性 销毁毒品。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 活动。了解这些“活性获得”突变的机制对于预测和 抑制它们的影响。 一条耐人寻味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。 也是一个全球性的临床祸害。鲍曼氏杆菌利用D类β-内酰胺酶OXA-24灭活青霉素类药物 和碳青霉烯类抗生素。最近，临床分离的具有扩大耐药性的鲍曼不动杆菌被追踪到 与底物识别相关的OXA-24柔性片段内的替换突变。这些 结果提出了构象动力学可以影响底物光谱的总体假设 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的柔韧性活性研究来研究这一假说 已经建立的替代突变体在临床上引起“活性增加”表型。我们的 研究使用液态核磁共振来表征游离酶和 底物和酰基-酶复合体，用于WT-OXA-24和耐药变异体。 目的1.比较载脂蛋白OXA-24/40及其临床变异体的构象样本。 目标2.确定络合物引起的配体构象灵活性的位点特异性变化 队形。 目的3.比较OXA-24/40/配体络合物及其配体的构象采样 临床变种。 对柔性蛋白质区域如何响应抗药性扩展突变的预测性理解 仍然是一个悬而未决的挑战。我们建议的研究通过调查以下因素来回答这个挑战 扩大革兰氏阴性抗生素耐药性的蛋白质灵活性。我们的结果可能会提出新的策略 为了改进抑制剂，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.