Conformational Flexibility and Antibiotic Resistance

构象灵活性和抗生素耐药性

• 批准号: 7920269
• 负责人: JEFFREY W PENG
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-24 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7920269
• 关键词: Acceleration; Accounting; Acylation; Affect; Affinity; Amino Acids; Anti-Infective Agents; Antibiotic Resistance; Antibiotics; Automobile Driving; Binding; Carbapenems; Cephalosporins; Clinic; Clinical; Containment; Data; Dissection; Drug resistance; Event; Exposure to; Failure; Goals; Investigation; Lactamase; Lactams; Length; Ligands; Light; Link; Mass Spectrum Analysis; Membrane; Methods; Molecular; Molecular Conformation; Monobactams; Multi-Drug Resistance; Multienzyme Complexes; Nosocomial Infections; Nuclear Magnetic Resonance; Pattern; Penicillins; Peptides; Phage Display; Principal Investigator; Production; Protein Dynamics; Proteins; Public Health; Resistance; Resolution; Roentgen Rays; Science; Scourge; Sensory; Series; Signal Transduction; Site; Solutions; Structure; Transducers; Work; base; combat; conformational conversion; coping; drug resistant bacteria; extracellular; flexibility; inhibitor/antagonist; methicillin resistant Staphylococcus aureus; pathogen; programs; protein structure; public health relevance; receptor; resistance mechanism; sensor; tool

DESCRIPTION (provided by applicant): Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant bacteria pathogen that has become a global clinical threat, accounting for nearly one-third of hospital-acquired infections. The MRSA threat is especially insidious due to its resistance to 2-lactam antibiotics (e.g. penicillins, carbapenems, cephalosporins), which remain the most widely used anti-infectives in the clinic. Elucidating the molecular basis for MRSA drug resistance is therefore imperative to develop strategies for its containment. A principal resistance mechanism of MRSA is the production of a ?-lactamase that hydrolytically destroys ?-lactam antibiotics. This production is triggered by the transmembrane sensor/transducer protein BlaR1. Exposure to a ?-lactam antibiotic triggers signal transduction by BlaR1, which leads to ?-lactamase production. Recent CD and IR studies demonstrate that the signal transduction entails conformational transitions in the extracellular sensor domain of BlaR1 (BlaRS henceforth) upon its binding of ?-lactam antibiotics. Defining these conformational transitions at the atomic level has become a critical goal in efforts to elucidate the signal transduction mechanism. Despite the availability of high-resolution protein structures, the atomic-level mechanism for the conformational transitions driving BlaR1 signal transduction remains obscure. We have begun studies of BlaRS, and our preliminary results point to a new set of molecular factors important for the BlaRS conformational transitions: protein conformational dynamics. Specifically, using solution Nuclear Magnetic Resonance (NMR), we observe that ?-lactam binding causes site-specific changes in local BlaRS flexibility. Hence, to elucidate the key events underlying BlaR1 signal transduction, we will investigate the functional consequences of BlaRS conformational dynamics. Accordingly, we propose three Specific Aims that will define how the interplay between protein dynamics and conformational change facilitates signal transduction on the part of BlaRS. These Aims include: (i) Comparing the site-specific changes in BlaRS flexibility upon activation by a series of different ?-lactam substrates; (ii) Defining the mechanism of interaction between BlaRS, and a peptide derived from the extra-cellular Loop 2 of the trans-membrane region of the BlaR1 receptor; (iii) Compare the effects of non-??-lactam inhibitors of BlaR1 versus ?-lactam substrates (antibiotics), on the dynamics and conformation of BlaRS. Our main experimental tool will be multi-dimensional NMR, which provides a uniquely powerful method for the atomic-level description of protein dynamics on a per- amino-acid-residue basis. Our studies will shed new light on the inner-workings of the signal transduction mechanism responsible for pernicious ?-lactam antibiotic resistance in MRSA. PUBLIC HEALTH RELEVANCE: This proposal describes studies to elucidate the molecular mechanisms whereby the sensor transducer protein, BlaR1, facilitates antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA), currently a global clinical scourge. The results will advance our abilities to cope with acceleration of multi-drug resistance in MRSA and other bacterial pathogens.

描述（由申请人提供）：耐甲氧西林金黄色葡萄球菌（MRSA）是一种多重耐药细菌病原体，已成为全球性的临床威胁，占医院获得性感染的近三分之一。 MRSA 的威胁尤其隐蔽，因为它对 2-内酰胺类抗生素（例如青霉素、碳青霉烯类、头孢菌素）具有耐药性，而这些抗生素仍然是临床上最广泛使用的抗感染药物。因此，阐明 MRSA 耐药性的分子基础对于制定遏制策略至关重要。 MRSA 的主要耐药机制是产生水解破坏 β-内酰胺抗生素的 β-内酰胺酶。这种产生是由跨膜传感器/转导蛋白 BlaR1 触发的。接触 β-内酰胺抗生素会触发 BlaR1 的信号转导，从而导致 β-内酰胺酶的产生。最近的CD和IR研究表明，信号转导需要BlaR1（以下简称BlaRS）的细胞外传感器结构域在与β-内酰胺抗生素结合时发生构象转变。在原子水平上定义这些构象转变已成为阐明信号转导机制的一个关键目标。尽管可以获得高分辨率的蛋白质结构，但驱动 BlaR1 信号转导的构象转变的原子级机制仍然不清楚。我们已经开始对 BlaRS 的研究，我们的初步结果指出了一组对 BlaRS 构象转变重要的新分子因素：蛋白质构象动力学。具体来说，使用溶液核磁共振（NMR），我们观察到β-内酰胺结合导致局部 BlaRS 灵活性发生位点特异性变化。因此，为了阐明 BlaR1 信号转导的关键事件，我们将研究 BlaRS 构象动力学的功能后果。因此，我们提出了三个具体目标，这些目标将定义蛋白质动力学和构象变化之间的相互作用如何促进 BlaRS 的信号转导。这些目标包括： (i) 比较一系列不同 β-内酰胺底物激活后 BlaRS 灵活性的位点特异性变化； (ii) 定义 BlaRS 和源自 BlaR1 受体跨膜区胞外环 2 的肽之间的相互作用机制； (iii)比较BlaR1的非β-内酰胺抑制剂与β-内酰胺底物（抗生素）对BlaRS的动力学和构象的影响。我们的主要实验工具将是多维核磁共振，它为基于氨基酸残基的蛋白质动力学原子级描述提供了一种独特的强大方法。我们的研究将为 MRSA 中导致有害的 β-内酰胺抗生素耐药性的信号转导机制的内部运作提供新的线索。公共健康相关性：本提案描述了旨在阐明传感器转导蛋白 BlaR1 促进耐甲氧西林金黄色葡萄球菌 (MRSA) 产生抗生素耐药性的分子机制的研究，该菌目前是全球性的临床祸害。这些结果将提高我们应对 MRSA 和其他细菌病原体的多重耐药性加速的能力。