TYPE IV PILI: STRUCTURAL ASPECTS OF THE ASSEMBLY MECHANI

IV 型 PILI：装配机械的结构方面

• 批准号: 6754412
• 负责人: Katrina T Forest
• 金额: $ 23.04万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6754412
• 关键词: Pseudomonas aeruginosa; X ray crystallography; bacteria; cell motility; molecular assembly /self assembly; pilin; posttranslational modifications; protein biosynthesis; protein structure; site directed mutagenesis; structural biology; thermophilic organism; virulence

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.

描述（改编自申请人的摘要）：许多致病性 微生物具有 IV 型菌毛，可以附着并定殖真核宿主 细胞进行毒性感染。 IV 型菌毛是长丝状细胞器 由数千个pilin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 射线晶体结构来确定毒力因子 菌毛生物发生蛋白。这些信息最终将用于 设计阻断组装、真核细胞结合的抑制剂，和/或 通过菌毛发出信号，从而充当抗生素。尤其是在这期间 将完成资金周期结晶和 X 射线结构确定 用于铜绿假单胞菌的菌毛蛋白亚基和菌毛 来自超嗜热菌 Aquifex aeolicus 的组装/运动因子 PilT。 需要 pilin 的亚基结构来检验 3-D 的假设 IV 型菌毛的亚基结构和寡聚堆积在整个过程中是保守的 物种。它还将揭示翻译后修饰。的结构 pilin 子单元将用于对组装好的子单元触点进行建模 菌毛纤维。结合起来，亚基结构和寡聚物模型将是 杂化菌毛蛋白分子设计的基础，以检验有关哪些部分的假设 菌毛蛋白分子之间以及与其他分子之间必须特异性地相互作用 生物发生机制中的蛋白质。 菌毛的两个主要作用是细菌和细菌之间的抽搐运动和信号传导 真核细胞需要 PilT 蛋白。 PilT 结构将揭示 PilT 表面可能与生物发生途径中的其他蛋白质相互作用。 将制备逻辑定点突变体来测试这些突变体的体内作用 表面。关键功能残基、效应子结合位点 预期的核苷酸结合口袋和可能的磷酸化位点将 进而被识别。连同可用的生化和遗传 数据，这些结构结果最终将产生高分辨率模型 菌毛生物发生和功能的分子机制。这 理解将是阻断pilus功能控制的基础 微生物感染。