Structure of Proteins Involved in Bacterial Pathogenesis

描述（由申请人提供）：革兰氏阴性菌的许多粘附素被掺入通过伴侣/引导途径组装的杂聚纤维中。这些纤维在形态上的范围从由伴侣蛋白的FGS（F1 G1短）亚家族成员组装的复合皮利到由伴侣蛋白的FGL（F1 G1长）亚家族组装的纤维囊状结构。在这里，我们建议建立在我们的工作P和1型皮利，这提供了一个范例，数百毒力纤维在不同的革兰氏阴性菌的组装。利用生物化学，遗传学，电子显微镜和X射线晶体学，我们已经确定了周质分子伴侣的结构和作用机制的显着细节。我们已经解决了复杂的尖端接头亚基，PapK，并与主要的尖端亚基，PapE的PapD周质伴侣的结构。我们还解决了复合物的PapE亚基的结构与N-末端延伸的PapK和结构的受体结合结构域的PapG粘附素有和没有其主机碳水化合物配体。菌毛分子伴侣由两个免疫球蛋白（IG）样结构域组成。这些结构表明菌毛蛋白亚基也具有Ig样折叠，但它们缺少其第七（G）链，从而暴露疏水核心。在我们称之为供体链互补的过程中，伴侣蛋白的G1链作为菌毛蛋白的第七链，催化亚基的折叠并防止非生产性亚基聚集。在引导器处，菌毛组装通过供体链交换发生，其中伴侣蛋白的G1链被存在于每个亚基上并暴露在进入的伴侣蛋白-亚基复合物上的N-末端延伸取代。菌毛亚基随后经历拓扑转变，触发其沟的闭合，将其邻居的NTE作为其自身IG折叠的一部分。因此，最终的菌毛结构是一系列Ig样结构域，每个结构域由两个单独的亚基单体的部分形成。每个亚基对其他相互作用的亚基具有不同的特异性，并且在菌毛组装中具有不同的作用。我们建议扩大我们的知识的结构的菌毛粘附素和进一步研究的结构的亚基和装配机械的两个这样的装配系统：原型FGS P菌毛系统的尿路致病性大肠杆菌和Saf FGL系统的沙门氏菌。此外，我们将进行抗分子伴侣化合物的结构研究。

DESCRIPTION (provided by applicant): Many of the adhesins of Gram-negative bacteria are incorporated into heteropolymeric fibers assembled by the chaperone/usher pathway. These fibers range in morphology from composite pili assembled by members of the FGS (F1G1 short) subfamily of chaperones, to fibrous capsule-like structures assembled by the FGL (F1G1 long) subfamily of chaperones. Here we propose to build on our work on P and type 1 pili, which has provided a paradigm for the assembly of hundreds of virulence fibers in diverse Gram-negative organisms. Using biochemistry, genetics, electron microscopy and X-ray crystallography, we have defined the structure and mechanism of action of the periplasmic chaperones in significant detail. We have solved the structure of the PapD periplasmic chaperone in complex with the tip adaptor subunit, PapK, and with the major tip subunit, PapE. We have also solved the structure of the PapE subunit in complex with the N-terminal extension of PapK and the structure of the receptor binding domain of the PapG adhesin with and without its host carbohydrate ligand. Pilus chaperones are comprised of two immunoglobulin (Ig)-like domains. These structures showed that pilin subunits also have an Ig-like fold, but they are missing their seventh (G) strand, thus exposing the hydrophobic core. In a process we termed donor-strand complementation, the chaperone's G1 strand serves as the pilin's seventh strand, catalyzing the folding of the subunit and preventing non-productive subunit aggregation. At the usher, pilus assembly occurs by donor-strand exchange, in which the G1 strand of the chaperone is replaced by an N-terminal extension that is present on every subunit and exposed on incoming chaperone-subunit complexes. The pilus subunit then undergoes a topological transition that triggers the closure of its groove, cementing its neighbor's NTE as part of its own Ig fold. Thus, the final pilus structure is a series of Ig-like domains, each of which is formed from parts of two individual subunit monomers. Each subunit has distinct specificity for other interactive subunits and distinct roles in pilus assembly. We propose to expand our knowledge of the structures of the pilus adhesins and further study the structure of the subunits and assembly machinery of two such assembly systems: the prototypical FGS P pilus system of uropathogenic Escherichia coli and the Saf FGL system of Salmonella. In addition, we will perform structural studies on anti-chaperone compounds.