Structure and Assembly of Type IV Pili & Related Systems

描述（由申请人提供）：对细菌毒力因子的分子理解对于解决对日益危险的抗生素耐药性微生物、新兴疾病和生物恐怖主义威胁的有效新疫苗和疗法的迫切需求至关重要。IV型药丸（T4 P）是革兰氏阴性菌的关键毒力因子，在表面运动、粘附、小菌落和生物膜形成、信号转导和DNA转化中具有不同的作用。这些表面暴露的T4 P是宿主免疫应答以及疫苗和治疗试剂的靶标。T4 P系统在结构上与II型分泌系统（T2 SS）相关，其在致病细菌物种中输出毒素，并且与运动所需的古生菌鞭毛系统相关。这三个系统采用多种蛋白质组分来构建跨越周质空间的多功能细丝。为了实现这种具有挑战性的细丝系统的详细表征，我们将通过X射线晶体学和电子显微镜整合生物化学，生物物理学和结构表征。这些实验的目的是解决一组原型的亚基和细丝结构，并确定装配ATP酶构象和它们的复合物与膜蛋白伴侣。我们将工作从亚基核心的构象状态和相互作用域，然后表征接口和蛋白质：蛋白质复合物。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T4 P和T2 SS系统的综合可视化，从而填补原核丝状组装体结构生物学中的关键空白。组装结构将通过诱变和氘交换质谱法进一步测试。因此，拟议的实验将建立一个分子描述的组件相关的理解其致病性变异逃避免疫反应，不同的生物学作用，药物发现的靶点，并设计与疫苗的保护性表位。

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.