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纳入通过伴侣/usher途径组装的异聚纤维中。这些纤维的形态范围从由伴侣蛋白的FGS （F1G1短）亚家族成员组装的复合毛，到由伴侣蛋白的FGL （F1G1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对P型和1型菌毛的研究基础上，这为不同革兰氏阴性菌中数百种毒力纤维的组装提供了范例。利用生物化学、遗传学、电子显微镜和x射线晶体学，我们详细地定义了质周伴侣蛋白的结构和作用机制。我们已经解决了PapD与尖端接头亚基（PapK）和主要尖端亚基（PapE）的周质伴侣复合物的结构。我们还解决了与PapK的n端延伸复合物中PapE亚基的结构，以及与宿主碳水化合物配体和不含宿主碳水化合物配体的PapG粘附素的受体结合域的结构。毛杆菌伴侣蛋白由两个免疫球蛋白（Ig）样结构域组成。这些结构表明，毛蛋白亚基也有一个类似于igg的折叠，但它们缺少第七股(G)链，从而暴露出疏水核心。在我们称为供体-链互补的过程中，伴侣G1链作为蛋白柱的第七链，催化亚基的折叠并防止非生产性亚基聚集。在usher中，毛组装通过供体链交换发生，其中伴侣的G1链被存在于每个亚基上的n端延伸所取代，并暴露在进入的伴侣-亚基复合物上。然后，毛状亚基经历一个拓扑转变，触发其凹槽的关闭，将其相邻的NTE巩固为其自身Ig褶皱的一部分。因此，最终的菌毛结构是一系列类似于igg的结构域，每个结构域都是由两个单独的亚基单体组成的。每个亚基对其他相互作用的亚基有不同的特异性，在菌毛组装中有不同的作用。我们建议扩大我们对菌毛粘附素结构的认识，并进一步研究两个这样的组装系统的亚基结构和组装机制：尿路致病性大肠杆菌的原型FGS - P菌毛系统和沙门氏菌的Saf - FGL系统。此外，我们将进行抗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.