TYPE IV PILI: STRUCTURAL ASPECTS OF THE ASSEMBLY

描述（改编自申请人的摘要）：许多致病性 微生物具有IV型皮利以附着和定殖真核宿主 细胞进行毒性感染。IV型皮利是长丝状细胞器 由数千个菌毛蛋白亚基拷贝组成。长期目标 这项工作的目的是了解这种分子组装机制， 通过解析IV型菌毛蛋白的X射线晶体结构， 菌毛生物发生蛋白。这些信息最终将用于 设计阻断组装、真核细胞结合和/或 通过皮利进行信号传导，从而用作抗生素。特别是在此期间， 将完成资助周期结晶和X射线结构测定 对于来自铜绿假单胞菌的菌毛蛋白亚基和菌毛， 来自超嗜热菌风产液菌的组装/运动因子PilT。 需要菌毛蛋白的亚基结构来检验3-D IV型皮利的亚基结构和寡聚体包装在不同的细胞中是保守的， 物种它还将揭示翻译后修饰。的结构 菌毛蛋白亚基将用于模拟组装的 菌毛纤维结合起来，亚基结构和寡聚体模型将是 设计杂交菌毛蛋白分子的基础，以测试关于哪些部分 菌毛蛋白分子的相互作用必须是特异性的， 蛋白质在生物发生机制中的作用。 皮利的两个主要作用，抽搐运动和细菌之间的信号传导， 真核细胞需要PilT蛋白。PilT结构将揭示 PilT表面可能与生物合成途径中的其他蛋白质相互作用。 将进行合理的定点突变以测试这些突变体的体内作用。 表面。关键功能残基，效应物结合位点，除了 预期的核苷酸结合口袋和可能的磷酸化位点将 此外，要识别。再加上现有的生化和遗传 数据，这些结构结果最终将导致高分辨率模型 菌毛生物发生和功能的分子机制。这 理解将是阻断菌毛功能的基础， 被微生物感染。

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.