Structure and binding studies of the bi-functional Chlamydia trachomatis protein

描述(申请人提供)：双功能沙眼衣原体蛋白Ct663在调节感染性基本体(EBS)和活跃分裂网状小体(RBS)之间的基本发育周期中的作用在分子水平上还不清楚。为Ct663的S功能提出的模型是并发的双重活性，这表明Ct663作为III型分泌系统伴侣和依赖于RNA聚合酶s66的转录抑制物的化学计量控制的调节机制。由于Ct663具有两种基本功能，是沙眼衣原体所特有的，是开发抗病毒药物的新靶点。为了开发针对沙眼衣原体发育周期的药物，首先必须确定控制Ct663‘S伴侣功能及其与蛋白伴侣相互作用的结构和热力学机制。拟议中的研究的长期目标是筛选和设计针对CT663的S功能的药物，这将防止细菌的传播；有可能直接治愈沙眼衣原体，如果与目前的治疗方法相结合，肯定还会减缓耐药菌株的发展。本应用的总体目标是建立一个CT663的模型，该模型包括CT663的三维高分辨率结构和CT663的S与伴侣Scc1和基本毒力因子CoPn的相互作用。中心假说是CT663的S化学计量比和络合物形成调控其双重活性。这项研究的基本原理是，干扰CT663的S相互作用和功能是一种有吸引力的治疗方法，因为它极有可能阻止细菌分化为感染性EB。这一应用的中心假设和目标将通过追求两个具体目标来检验和实现：(1)通过测定络合物的化学计量、解离常数和四元结构来建立(CT663：Scc1)：COPN组装的模型；(2)确定CT663的三维结构并确定其Scc1结合界面。预计这些目标将产生用于合理药物设计的Ct663及其络合物模型的预期结果。这一结果有望产生重要的积极影响，因为Ct663的结构和功能模型除了从根本上促进结构生物学和传染病生物学领域的发展外，还将为治疗干预提供新的靶点。这一贡献意义重大，因为这是一系列研究的第一步，预计将有助于了解CT663的S的双重功能，并有助于治疗最常见的性传播细菌疾病。在我看来，拟议的研究是创新的，因为它代表了对现状的新的和实质性的偏离，即利用核磁共振来靶向细菌毒力作为治疗策略的沙眼衣原体独特的双功能蛋白的结构和生化特征。

DESCRIPTION (provided by applicant): The role of the bi-functional Chlamydia trachomatis protein, Ct663, in regulating the essential, developmental cycle between infectious elementary bodies (EBs) and actively dividing, reticulate bodies (RBs) is not well-understood at the molecular level. The model proposed for Ct663's functions is concurrent dual activity, which suggests a regulatory mechanism involving stoichiometric control of Ct663 as a type III secretion system chaperone and an RNA polymerase s66-dependent transcriptional inhibitor. Because Ct663 has two essential functions and is unique to C. trachomatis, it is a novel target for developing anti- virulence drugs. In order to develop drugs targeting the developmental cycle of C. trachomatis, the struc- tural and thermodynamic mechanisms controlling Ct663's chaperone function and its interaction with its protein partners must first be determined. The long-term goal of the proposed research is to screen and design drugs targeting Ct663's functions, which would prevent the bacteria from spreading; likely curing C. trachomatis directly and certainly mitigating the development of antibiotic-resistant strains, when com- bined with current therapies. The overall objective of this application is to develop a model of Ct663 that includes the 3-dimensional high-resolution structure of Ct663 and characterization of Ct663's interactions with the chaperone, Scc1, and the essential virulence factor, CopN. The central hypothesis is that Ct663's stoichiometry and complex formation regulates its dual activity. The rationale for the proposed research is that disrupting Ct663's interactions and functions is an attractive therapy because it is highly likely to prevent the bacteria from differentiating into infectious EB's. The central hypothesis and objec- tive of this application will be tested and attained by pursuing two specific aims: (1) develop a model of (Ct663:Scc1):CopN assembly by determining the stoichiometry, dissociation constants, and quaternary structure of the complexes and (2) determine the 3D structure of Ct663 and identify its Scc1-binding in- terface. It is anticipated that these aims will yield the expected outcome of a model of Ct663 and its complexes for rational drug design. This outcome is expected to have an important positive impact be- cause a structural and functional model of Ct663 will provide a new target for therapeutic interventions in addition to 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 of Ct663's dual functions and contribute to the treatment of the most common, sexually transmitted bacterial disease. The proposed research is innovative, in my opinion, because it represents a new and substantive departure from the status quo, namely the structural and biochemical characteri- zation of a unique bi-functional protein from C. trachomatis using NMR to target bacterial virulence as a therapeutic strategy.