TYPE IV PILI: STRUCTURAL ASPECTS OF THE ASSEMBLY MECHANI

描述(改编自申请人的摘要)：许多致病因素 微生物有IV型菌毛附着并定植于真核宿主 用于致病性感染的细胞。IV型菌毛是长丝状细胞器 由数千个Pilin亚单位的拷贝组成。长期目标 这项工作的目的是了解这一分子组装机制 通过解算IV型菌毛蛋白的x射线晶体结构确定毒力因子 菌毛生物发生蛋白。这些信息最终将被用来 设计阻断组装、真核细胞结合和/或 通过菌毛发出信号，从而充当抗生素。尤其是在这个过程中， 将进行资金周期结晶和x射线结构测定。 铜绿假单胞菌和菌毛中的菌毛蛋白亚基 嗜热性Aquifex aeolicus的组装/运动因子PILT。 需要Pilin的亚基结构来检验这一假设 IV型菌毛的亚基结构和寡聚堆积是保守的 物种。它还将揭示翻译后的修改。的结构 Pilin亚单位将被用来模拟组装后的 毛发纤维。结合起来，亚基结构和低聚物模型将是 杂化毛细蛋白分子的设计基础，以检验关于哪些部分的假说 的分子必须相互作用，并且相互作用。 生物发生机制中的蛋白质。 菌毛的两个主要作用是在细菌和细菌之间的抽动运动和信号传递 对于真核细胞，需要PILT蛋白。PILT结构将揭示 PILT表面可能与生物发生途径中的其他蛋白质相互作用。 逻辑定点突变将被用来测试这些基因在体内的作用 表面。关键功能残基、效应器结合部位以及 预期的核苷酸结合口袋和可能的磷酸化位点将 此外，还可以确定。以及现有的生化和基因 数据，这些结构结果最终将导致高分辨率模型 菌毛的生物发生和功能的分子机制。这 理解将是阻止Pilus功能进行控制的基础 微生物感染。

DESCRIPTION (Adapted from the Applicant's Abstract): Many pathogenic microorganisms have Type IV pili to attach to and colonize eukaryotic host cells for virulent infection. The type IV pili are long filamentous organelles comprised of thousands of copies of the pilin subunit. The long term objective of this work is to understand the molecular assembly mechanism of this virulence factor by solving the x-ray crystal structures of type IV pilins and the pilus biogenesis proteins. This information will ultimately be used to design inhibitors that block assembly, eukaryotic cell binding, and/or signaling by pili, thereby serving as antibiotics. In particular, during this funding cycle crystallization and x-ray structure determination will be done for the pilin subunit from Pseudomonas aeruginosa and the pilus assembly/motility factor PilT from the hyperthermophile Aquifex aeolicus. The subunit structure of pilin is needed to test the hypothesis that the 3-D subunit structure and oligomeric packing of type IV pili is conserved across species. It will also reveal posttranslational modifications. The structure of the pilin subunit will be used to model the subunit contacts in an assembled pilus fiber. Combined, the subunit structure and oligomer model will be the basis for design of hybrid pilin molecules to test hypotheses about which parts of the pilin molecule must interact specifically with each other and with other proteins in the biogenesis machinery. Two main roles of pili, twitching motility and signaling among bacteria and with eukaryotic cells, require the PilT protein. The PilT structure will reveal PilT surfaces likely to interact with other proteins in the biogenesis pathway. Logical site-directed mutants will be made to test the in vivo roles of these surfaces. Critical functional residues, effector binding sites in addition to the expected nucleotide binding pocket, and possible phosphorylation sites will furthermore be identified. Together with available biochemical and genetic data, these structural results will eventually lead to a high resolution model of the molecular mechanisms of pilus biogenesis and function. This understanding will be the foundation for blocking pilus functions to control infection by microbes.