Allosteric adhesins of enterobacterial pathogens

肠杆菌病原体的变构粘附素

基本信息

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
项目类别：
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

项目摘要

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.

抽象的该建议旨在确定人类粘附的潜在变构特性肠杆菌病原体-Escherichia Coli，Klebsiella肺炎/oxytoca，肠杆菌属，proteus Mirabilis和沙门氏菌 - 通过伴侣糖途径（杯）组装。迄今为止，只有大肠杆菌的甘露糖特异性，1型纤维化粘附蛋白已被证明是一种变构蛋白这可以存在于替代功能（主动/非活动）构象中。该特性允许包含的细菌 FIMH作为头发状表面附属的一部分，即fimbriae或pili，以迅速结合宿主细胞上的配体从不活跃的构象中，在剪切力下持续很长的寿命。主动构象。长寿（缓慢解离）结合涉及形成所谓的“接管键” 在拉伸机械力下可以激活并变得更强壮，并涉及变构开关。到日期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中（主动/非活动）构象。为了确定通过类似机制起作用的其他依从性，我们将重点关注粘性是fimbriae或pili的一部分，属于与FIMH相同的杯子结构类别。我们最近确定了一组脂肪族或芳族保留，它们充当分子切换，控制变构开关通过在蛋白质核心和表面之间切换其方向之间的活性和无活性构象之间。可以通过“表面锁定”稳定粘合剂的活动或不活动构象通过取代为亲水带电残差。我们将使用FIMH切换的假定类似物在其他杯子粘附中确定变构状态的存在 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EM。我们的研究成功将有助于理解一般机制细菌对宿主细胞的粘附以及最终对优化疫苗和小分子的设计抑制剂。如果发现某些粘附剂是变构的，则对其物理相关的深度分析结构/功能特性以及发病机理的重要性以及实际实施发现将是未来研究的重点。