Structure and Assembly of Type IV Pili & Related Systems

描述(由申请人提供)：对细菌毒力因素的分子理解对于解决日益危险的抗生素耐药微生物、新出现的疾病和生物恐怖主义威胁的有效新疫苗和治疗方法的迫切需求至关重要。IV型药丸(T4P)是革兰氏阴性菌的关键毒力因子，在表面运动、黏附、微菌落和生物膜的形成、信号转导和DNA转化等方面发挥着不同的作用。这些暴露在表面的T4P是宿主免疫反应的靶标，也是疫苗和治疗试剂的靶标。T4P系统在结构上与II型分泌系统(T2SS)有关，后者输出致病细菌物种中的毒素，并与运动所需的古细菌鞭毛系统有关。这三个系统使用多个蛋白质组分来构建跨越周质空间的多功能细丝。为了对这种具有挑战性的细丝系统进行详细的表征，我们将通过X射线结晶学和电子显微镜将生化、生物物理和结构表征结合起来。这些实验旨在解决一组典型的亚基和细丝结构，并确定组装的ATPase构象及其与膜蛋白伙伴的复合体。我们将从亚基核心向外工作，直到构象状态和相互作用区域，然后表征界面和蛋白质：蛋白质复合体。因此，这一联合Tainer-Craig提案将提供从单个蛋白质结构域到复杂的T4P和T2SS系统的集成可视化，从而填补原核丝状组件结构生物学中的一个关键空白。组装结构将通过诱变和重离子交换质谱仪进行进一步测试。因此，拟议的实验将建立与了解其致病变异以逃避免疫反应、不同的生物学作用、药物发现的目标位置以及疫苗保护性表位设计相关的组件的分子描述。

DESCRIPTION (provided by applicant): A molecular understanding of bacterial virulence factors is critical to address the urgent need for effective new vaccines and therapeutics for increasingly dangerous antibiotic resistant microbes, emerging diseases and bio-terrorism threats. Type IV pill (T4P) are key virulence factors for Gram negative bacteria with diverse roles in surface motility, adhesion, microcolony and biofilm formation, signal transduction, and DNA transformation. These surface-exposed T4P are targets for the host immune response, as well as for vaccines and therapeutic reagents. The T4P system is structurally related to the Type II secretion system (T2SS), which exports toxins in pathogenic bacterial species, and to the archaeal flagellar system, needed for motility. These three systems employ multiple protein components to build multifunctional filaments spanning the periplasmic space. To achieve detailed characterizations of such challenging filament systems, we will integrate biochemical, biophysical and structural characterizations by both x-ray crystallography and electron microscopy. These experiments aim to solve a prototypical set of subunit and filament structures, and to define assembly ATPase conformations and their complexes with membrane protein partners. We will work outward from the subunit cores to the conformational states and interaction domains and then to characterize interfaces and protein:protein complexes. This joint Tainer-Craig proposal will thus provide an integrated visualization built up from the individual protein domains to the complex T4P and T2SS systems and thereby fill a critical gap in the structural biology of prokaryotic filamentous assemblies. Assembly structures will be further tested by mutagenesis and deuterium exchange-mass spectrometry. The proposed experiments will thus build a molecular description of assemblies relevant to understanding their pathogenic variation to escape the immune response, diverse biological roles, target sites for drug discovery, and design with protective epitopes for vaccines.