Conformation and functional dynamics of a bacterial PASTA kinase

项目摘要/摘要 粪肠球菌等肠球菌是数万亿种肠道微生物中通常共存于 与寄主的共生关系。然而，对抗生素具有抗药性的肠球菌也是最多的三种 这是医院获得性感染的常见原因，因此是一个严重的公共卫生问题。一个 医院获得性肠球菌感染的重要危险因素是事先接受过 肠球菌天生耐药的抗生素，如广谱头孢菌素。因此， 肠球菌固有的定植肠道和抵抗抗菌剂攻击的能力尤为突出。 对肠球菌感染的发病机制至关重要。然而，肠球菌肠道定植的机制和 对抗菌素的耐药性还没有完全了解。这些内在特征的一个关键决定因素是 跨膜型丝氨酸/苏氨酸蛋白激酶，又称Irek，属于细菌蛋白激酶家族，含有 细胞外的意大利面域。这种“面食蛋白激酶”控制着包括抗生素耐药性在内的关键过程， 几乎所有革兰氏阳性细菌中的毒素产生、毒力或细胞分裂；在某些细菌中，是一种面食酵素 对于生存来说是必不可少的。因此，意大利面激酶是新疗法的诱人靶点。然而，a 基本了解意大利面条蛋白在溶液中的结构和动力学，以及其结构和功能 缺乏促进环境感知和体内生物反应协调的关系。 这些信息对于开发新的治疗方法至关重要。在这里提出的这项研究 寻求通过阐明解决方案中的基本结构-功能关系来解决这一差距 具有代表性的意大利面激酶，来自粪肠球菌的Irek激酶，我们已经证明了它是内在的 粪肠球菌对细胞壁活性抗生素和胆汁中存在的洗涤剂的耐药性。这种协作性 研究旨在应用新的方法和视角来阐明对 体内功能分析和EPR波谱互补结合的Irek激酶 确定溶液中的意大利面模块构象和了解激活域的方法 动力学有助于激活剂的激活。通过圆满完成这项工作，我们将获得重要的新的 对具有代表性的细菌面蛋白激酶的洞察，以帮助确定其结构-功能关系 关键细菌信号蛋白的整个家族，其长期目标是调节这些蛋白的活性 治疗上的激活酶。

Project Summary/Abstract Enterococci such as Enterococcus faecalis are among the trillions of intestinal microbes that normally coexist in a symbiotic relationship with their host. However, antibiotic-resistant enterococci are also among the three most common causes of hospital-acquired infections and therefore represent a serious public health problem. An important risk factor for the acquisition of hospital-acquired enterococcal infections is prior therapy with antibiotics, such as broad-spectrum cephalosporins, to which enterococci are intrinsically resistant. Thus, the inherent abilities of enterococci to colonize the gut and withstand assault by antimicrobial agents are especially critical for pathogenesis of enterococcal infections. Yet, the mechanisms of enterococcal gut colonization and antimicrobial resistance are not fully understood. One critical determinant of these intrinsic traits is a transmembrane Ser/Thr kinase known as IreK, which belongs to a family of bacterial kinases containing extracellular PASTA domains. Such “PASTA kinases” control critical processes including antibiotic resistance, toxin production, virulence, or cell division in nearly all Gram-positive bacteria; in some bacteria, a PASTA kinase is essential for viability. As such, PASTA kinases represent attractive targets for new therapeutics. However, a basic understanding of the structure and dynamics of PASTA kinases in solution, and the structure-function relationships that promote environmental sensing and coordination of biological responses in vivo, is lacking. Such information is critical to inform development of new therapeutic approaches. The research proposed here seeks to address this gap by elucidating fundamental structure-function relationships in solution for a representative PASTA kinase, the IreK kinase from E. faecalis, which we have shown is required for intrinsic resistance of E. faecalis to cell-wall-active antibiotics and to detergents present in bile. This collaborative research is designed to apply new approaches and perspectives to elucidate novel insights into the function of the IreK kinase using a complementary combination of in vivo functional assays and EPR spectroscopy approaches to determine the PASTA module conformation in solution and understand how kinase domain dynamics contribute to kinase activation. By successfully completing this work, we will obtain important new insights into a representative bacterial PASTA kinase to help define the structure-function relationships for an entire family of critical bacterial signaling proteins with the long-term goal of modulating the activity of these kinases therapeutically.