Conformational Flexibility and Antibiotic Resistance

说明(申请人提供)：耐甲氧西林金黄色葡萄球菌(MRSA)是一种多药耐药细菌病原体，已成为全球临床威胁，占医院感染的近三分之一。MRSA的威胁尤其隐蔽，因为它对2-内酰胺类抗生素(如青霉素类、碳青霉烯类、头孢菌素类)的耐药性，这些抗生素仍然是临床上使用最广泛的抗感染药物。因此，阐明耐甲氧西林金黄色葡萄球菌耐药性的分子基础是制定遏制其耐药性的策略的当务之急。耐甲氧西林金黄色葡萄球菌的一个主要耐药机制是产生一种能水解性破坏β-内酰胺类抗生素的β-内酰胺酶。这种产生是由跨膜传感器/转导蛋白BlaR1触发的。暴露于β-内酰胺类抗生素会触发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，当BlaR1与β-内酰胺类抗生素结合时，信号转导需要在BlaR1(以下简称BlaRS)的胞外传感器区域发生构象转变。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关键目标。尽管已经有了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水平机制仍然不清楚。我们已经开始了对BLARS的研究，我们的初步结果指出了一组新的对BLARS构象转变重要的分子因素：蛋白质构象动力学。具体地说，利用溶液核磁共振(NMR)，我们观察到β-内酰胺结合引起局部BlaRS灵活性的位点特异性变化。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BLARS部分的信号转导。这些目的包括：(I)比较一系列不同的β-内酰胺底物激活时BlaRs灵活性的部位特异性变化；(Ii)确定BlaRs与BlaR1受体跨膜区细胞外环2衍生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，它为以每个氨基酸残基为基础的蛋白质动力学的原子水平描述提供了一种独特而强大的方法。我们的研究将进一步揭示耐甲氧西林金黄色葡萄球菌对β-内酰胺类抗生素耐药的信号转导机制的内部机制。公共卫生相关性：这项建议描述了一些研究，以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)耐药的分子机制，MRSA目前是全球临床疾病。这一结果将提高我们应对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.