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 这项建议旨在确定人类粘附素中潜在的变构性质 肠杆菌病原体--大肠埃希菌、肺炎克雷伯菌/催产素、肠杆菌属、变形杆菌 奇异杆菌和沙门氏菌--通过伴侣-引座者途径(CUP)组装。到目前为止，只有 大肠杆菌甘露糖特异的1型菌毛粘附素FimH已被证明是一种变构蛋白 可以以交替的功能(活性/非活性)构象存在。这一特性使细菌能够含有 FimH作为毛状表面附属物、菌毛或菌毛的一部分，快速结合寄主细胞上的配体 从不活跃的构象转变为在剪切力作用下保持很长寿命 活性构象。这种长期(缓慢解离)的结合包括形成所谓的“捕获键”。 它可以在拉伸机械压力下被激活并变得更强，并涉及变构开关。至 目前还没有其他细菌粘附素被证明是变构的，并且存在于替代功能 (活性/非活性)构象。为了确定其他通过类似机制起作用的粘附素，我们将重点放在 属于菌毛或菌毛的一部分，与FimH属于同一杯状结构类别的粘附素。我们最近 确定了一组作为控制变构开关的分子开关的脂肪族或芳香族残基 通过在蛋白质核心和表面之间切换它们的方向来区分活跃和不活跃的构象。 可以通过这样的切换来稳定粘附素的活性或非活性构象 通过取代到亲水的带电残基。我们将使用假定的FimH切换的类比来 鉴定其他杯状粘附素中同源或非同源的变构状态的存在 FimH，使用诱变，各种功能分析和三种类型的结构分析--核磁共振，X射线 结晶学和低温电子显微镜。我们研究的成功将有助于对一般机制的理解 细菌与宿主细胞的粘附性，最终与优化疫苗和小分子的设计有关 抑制剂。如果发现某些粘附素是变构的，深入分析它们的生理相关性 结构/功能特性及其在发病机制和实际应用中的意义 这些发现将是未来研究的重点。