Conformational Flexibility and Antibiotic Resistance

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，已成为全球临床威胁，占医院获得性感染的近三分之一。MRSA的威胁是特别阴险的，因为它对2-内酰胺类抗生素（例如青霉素类、碳青霉烯类、头孢菌素类）具有抗性，2-内酰胺类抗生素仍然是临床上最广泛使用的抗感染药。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一种？水解破坏的内酰胺酶？内酰胺类抗生素这种生产是由跨膜传感器/转导蛋白BlaR 1触发的。暴露于？-内酰胺类抗生素通过BlaR 1触发信号转导，从而导致？-内酰胺酶产生。最近的CD和IR研究表明，信号转导需要在BlaR 1的细胞外传感器结构域（BlaRS）中的构象转变，其结合？内酰胺类抗生素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关键目标。尽管高分辨率的蛋白质结构的可用性，驱动BlaR 1信号转导的构象转变的原子水平的机制仍然不清楚。我们已经开始研究的BlaRS，我们的初步结果指向一组新的分子因素的重要BlaRS构象转变：蛋白质构象动力学。具体地说，使用溶液核磁共振（NMR），我们观察到？-内酰胺结合导致局部BlaRS柔性的位点特异性变化。因此，为了阐明BlaR 1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的目标，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信号转导的BlaRS的一部分。这些目的包括：（i）比较BlaRS灵活性在一系列不同的？内酰胺底物;（ii）确定BlaRS与来自BlaR 1受体跨膜区细胞外环2的肽之间的相互作用机制;（iii）比较非？？- BlaR 1的内酰胺抑制剂与？-内酰胺底物（抗生素），对BlaRS的动力学和构象。我们的主要实验工具将是多维NMR，它提供了一个独特的强大的方法，原子水平上的蛋白质动力学描述的每氨基酸残基的基础上。我们的研究将为恶性肿瘤的信号转导机制的内部运作提供新的线索。耐甲氧西林金黄色葡萄球菌内酰胺类抗生素耐药性。公共卫生相关性：该提案描述了阐明传感器转导蛋白BlaR 1促进耐甲氧西林金黄色葡萄球菌（MRSA）抗生素耐药性的分子机制的研究，该耐甲氧西林金黄色葡萄球菌目前是全球临床灾难。这些结果将提高我们科普MRSA和其他细菌病原体多重耐药性加速的能力。

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.