Allosteric adhesins of enterobacterial pathogens

肠杆菌病原体的变构粘附素

• 批准号: 10512013
• 负责人: Rachel E Klevit
• 金额: $ 72.74万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-24 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10512013
• 关键词: Adhesions; Adhesives; Affinity; Amino Acids; Bacteria; Bacterial Adhesins; Bacterial Adhesion; Binding; Binding Proteins; Biological Assay; Cell Adhesion; Cell surface; Cells; Characteristics; Charge; Core Protein; Cryoelectron Microscopy; Crystallography; Dissociation; Enterobacter; Enterobacteriaceae; Escherichia coli; Eukaryota; Family; Fimbriae Proteins; Fimbrial Adhesins; Future; Goals; Hair; Human; Image; Intuition; Kinetics; Klebsiella pneumoniae; Knowledge; Lead; Lectin; Ligand Binding; Ligands; Mannose; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Multi-Drug Resistance; Mutagenesis; Mutation; Oligosaccharides; Pathogenesis; Pathway interactions; Physiological; Pilum; Positioning Attribute; Property; Protein Dynamics; Proteins; Proteus mirabilis; Receptor Cell; Rod; Salmonella; Shapes; Side; Solvents; Structure; Surface; Testing; Tissues; Uropathogenic E. coli; Vaccines; Virulence Factors; Work; X-Ray Crystallography; analog; appendage; base; conformational conversion; conformer; design; fimbria; human pathogen; hydrophilicity; insight; mechanical force; molecular dynamics; mutant; pathogen; pathogenic bacteria; pragmatic implementation; protein structure; shear stress; small molecule inhibitor; success; sugar; tool

Abstract This proposal seeks to identify potential allosteric properties in adhesins of human enterobacterial pathogens - Escherichia coli, Klebsiella pneumoniae/oxytoca, Enterobacter spp, Proteus mirabilis, and Salmonella – that are assembled via a chaperone-usher pathway (CUP). To date, only the mannose-specific, type 1 fimbrial adhesin of E. coli, FimH, has been demonstrated to be an allosteric protein that can exist in alternative functional (active/inactive) conformations. This property allows bacteria that contain FimH as part of hair-like surface appendages, fimbriae or pili, to bind ligand presented on host cells rapidly from an inactive conformation and to remain bound for very long lifetimes under shear force by transiting to an active conformation. The long-lived (slow dissociation) binding involves formation of so-called `catch-bonds' that can be activated and become stronger under tensile mechanical force and involve an allosteric switch. To date no other bacterial adhesin has been demonstrated to be allosteric and to exist in alternative functional (active/inactive) conformations. To identify other adhesins that work via similar mechanisms, we will focus on adhesins that are part of fimbriae or pili and belong to the same CUP structural class as FimH. We recently identified a set of aliphatic or aromatic residues that act as molecular toggles that control the allosteric switch between active and inactive conformations by switching their orientation between the protein core and surface. It is possible to stabilize either active or inactive conformation of the adhesin by “surface locking” such toggles through substitution to hydrophilic charged residues. We will use putative analogs of the FimH toggles to identify the existence of allosteric states in other CUP adhesins that are homologous or non-homologous to FimH, using mutagenesis, various functional assays, and three types of structural analysis – NMR, X-ray crystallography, and cryo-EM. Success of our studies will contribute to understanding of general mechanisms of bacterial adhesion to host cells and, ultimately, to the design of optimized vaccines and small molecule inhibitors. If certain adhesins are found to be allosteric, in-depth analysis of their physiologically-relevant structure/functional properties and significance for pathogenesis as well as practical implementation of the findings will be the focus of future studies.

摘要 该建议旨在鉴定人粘附素中潜在的变构性质， 肠细菌病原体-大肠埃希菌、肺炎克雷伯菌/产酸克雷伯菌、肠杆菌属、变形杆菌 奇异菌和沙门氏菌-它们通过伴侣引导途径（CUP）组装。到目前为止，只有 甘露糖特异的E. FimH是一种变构蛋白 可以存在于替代的功能（活性/非活性）构象中。这种特性使得含有 FimH作为毛发样表面附属物（菌毛或皮利）的一部分，快速结合宿主细胞上呈递的配体 从非活性构象，并在剪切力下通过转变为 活性构象长寿命（缓慢解离）结合涉及形成所谓的“捕获键” 在拉伸机械力下可以被激活并变得更强，并涉及变构开关。到 迄今为止，没有其他细菌粘附素被证明是变构的，并且以替代功能存在。 （活性/非活性）构象。为了鉴定通过类似机制起作用的其他粘附素，我们将重点关注 粘附素是菌毛或皮利的一部分，并且与FimH属于相同的CUP结构类别。我们最近 确定了一组脂肪族或芳香族残基，它们作为控制变构开关的分子开关 通过在蛋白质核心和表面之间转换它们的方向，在活性和非活性构象之间进行转换。 通过“表面锁定”，例如触发器， 通过取代成亲水性带电残基。我们将使用FimH切换的假定类似物， 鉴定与CUP粘附素同源或非同源的其他CUP粘附素中变构状态的存在， FimH，使用诱变，各种功能测定和三种类型的结构分析- NMR，X射线 晶体学和冷冻电镜我们研究的成功将有助于理解一般机制 细菌粘附宿主细胞，并最终设计优化的疫苗和小分子 抑制剂的如果发现某些粘附素是变构的，深入分析其生理相关性， 结构/功能特性和发病机制的意义以及实际实施的 研究结果将是未来研究的重点。