Protein-protein interactions and structural switching of the bifunctional Chlamydia trachomatis protein, Scc4

项目摘要 双功能沙眼衣原体蛋白Scc 4（以前称为Ct 663）在调节III型se-DNA中的作用， 分泌系统（T3 SS）和基本的发育周期（感染性基本体和感染性基本体之间）， 在分子水平上还没有很好地理解。Scc 4是一种独特的蛋白质， （i）作为T3 SS分子伴侣与Scc 1一起调节CopN（一种必需的毒力因子）的分泌， (ii)作为转录因子调节β 66-RNA聚合酶全酶。根据2010年期间取得的进展， 上一个资助期，为Scc 4的双重功能提出的转换机制涉及一个构象 改变其三维结构。由于Scc 4具有两个基本功能，并且是C语言所独有的。粗的，粗的 因此，它是开发抗病毒药物的新靶点。开发针对C.沙眼衣原体 T3 SS和发育周期，必须首先鉴定命中化合物及其对Scc 4结构的影响， 蛋白质-蛋白质相互作用和功能特征。这项研究的长期目标是： 针对Scc 4功能的药物，这将阻止细菌传播，可能治愈C。反，反 chomatis直接和肯定减轻耐药菌株的发展，当与 目前的治疗。这个应用程序的总体目标是增加我们对共形的理解- 调节Scc 4的开关机制和蛋白质-蛋白质相互作用的分子变化和结合位点。 中心假设是Scc 4结构中的构象转换，由结合天然配体诱导， 调节其双重活动。这项研究的基本原理是， 可以合理设计，从而为治疗C.沙眼和新的分子工具， 研究T3 SS和发育周期。本申请的中心假设和目标将是 通过追求两个特定目标来测试和实现：（1）鉴定结合或破坏Scc 4的小分子配体 及其T3 SS复合物，以及（2）绘制Scc 4上的天然蛋白质-和小分子-配体结合位点， Scc 4：Scc 1并鉴定变构效应。预计这些目标将产生预期的成果， Scc 4上的配体结合位点的结构模型和来自天然配体和hit-com的Scc 4：Scc 1复合物， 磅这一结果预计将产生重要的积极影响，因为蛋白质的表征 结合位点和小分子配体的鉴定提供了开发药物所必需的信息 靶向Scc 4的构象开关或蛋白质-蛋白质相互作用，从根本上推进了 结构生物学和传染病生物学。这一贡献意义重大，因为它是一个 一系列研究，预计将导致理解Scc 4在原子水平上的双重功能， 有助于治疗最常见的性传播细菌疾病。拟议研究 是创新的，因为独特的双功能蛋白质的NMR结构和生物化学表征 梭沙眼衣原体靶向细菌毒力作为治疗策略代表了一种新的和实质性的 脱离现状。

PROJECT SUMMARY The role of the bi-functional Chlamydia trachomatis protein, Scc4 (formerly Ct663), in regulating the type III se- cretion system (T3SS) and the essential, developmental cycle (between infectious elementary bodies and ac- tively dividing, reticulate bodies) is not well-understood at the molecular level. Scc4 is a unique protein that functions (i) as a T3SS chaperone with Scc1 to regulate secretion of CopN (an essential virulence factor) and (ii) as a transcription factor regulating 66-RNA polymerase holoenzyme. Based on progress made during the last funding period, the switching mechanism proposed for Scc4’s dual functions involves a conformational change in its 3-dimensional structure. Because Scc4 has two essential functions and is unique to C. tracho- matis, it is a novel target for developing anti-virulence drugs. To develop drugs targeting the C. trachomatis T3SS and developmental cycle, hit-compounds must first be identified and their effects on Scc4’s structure, protein-protein interactions, and functions characterized. The long-term goal of the proposed research is to de- velop drugs targeting Scc4’s functions, which would prevent the bacteria from spreading, likely curing C. tra- chomatis directly and certainly mitigating the development of antibiotic-resistant strains, when combined with current therapies. The overall objectives of this application are to increase our understanding of the conforma- tional changes and binding sites that regulate Scc4’s switching mechanism and protein-protein interactions. The central hypothesis is that a conformational switch in Scc4’s structure, induced by binding native ligands, regulates its dual activity. The rationale for the proposed research is that inhibitors to disrupt Scc4’s functions can be rationally designed, resulting in new approaches to treat C. trachomatis and new molecular tools to study the T3SS and developmental cycle. The central hypothesis and objectives of this application will be tested and attained by pursuing two specific aims: (1) identify small molecule ligands that bind or disrupt Scc4 and its T3SS complexes and (2) map the native protein- and small molecule-ligand binding sites on Scc4 and Scc4:Scc1 and identify allosteric effects. It is anticipated that these aims will yield the expected outcome of a structural model of ligand-binding sites on Scc4 and the Scc4:Scc1 complex from native ligands and hit-com- pounds. This outcome is expected to have an important positive impact because characterization of the protein binding sites and identification of small molecule ligands provide the information necessary to develop drugs targeting Scc4’s conformational switch or protein-protein interactions, fundamentally advancing the fields of structural biology and infectious disease biology. This contribution is significant because it is the first step in a continuum of research that is expected to lead to understanding Scc4’s dual functions at the atomic level and contribute to the treatment of the most common, sexually transmitted bacterial disease. The proposed research is innovative because the NMR structural and biochemical characterization of a unique bi-functional protein from C. trachomatis to target bacterial virulence as a therapeutic strategy represents a new and substantive departure from the status quo.