The interaction between outer membrane porins and toll-like receptors

外膜孔蛋白与Toll样受体之间的相互作用

• 批准号: 8144343
• 负责人: T M Iverson
• 金额: $ 19.31万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-16 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8144343
• 关键词: Acetylation; Affect; Affinity; Area; Autoimmune Diseases; Bacteria; Binding; Biological; Caliber; Charge; Chemicals; Complex; Crystallography; Data; Development; Electron Microscopy; Electrons; Electrostatics; Elements; Event; Experimental Designs; Exploratory/Developmental Grant; Funding; Goals; Grant; Haemophilus influenzae; Homo; Hybrids; Immune; Immune response; Immune system; In Vitro; Inflammatory Response; Invaded; Lead; Ligands; Literature; Location; Lysine; Maps; Membrane; Membrane Proteins; Meningitis; Methods; Methylation; Modification; Molecular; Natural Immunity; Neisseria meningitidis; Physiological; Process; Protein Binding; Proteins; Protozoa; Publishing; Reporting; Research; Research Project Grants; Resolution; Risk; Scanning; Side; Signal Transduction; Sodium Chloride; Staging; Structure; Surface; System; TLR1 gene; TLR2 gene; TLR4 gene; TLR6 gene; Techniques; Testing; Toll-like receptors; Virus; Vision; Work; base; dimer; fungus; in vivo; particle; pathogen; pathogenic bacteria; physical property; porin; public health relevance; receptor; receptor binding; reconstruction; research study; response

DESCRIPTION (provided by applicant): In the innate immune system, Toll-like receptors (TLRs) provide a front-line defense against invading bacteria, viruses, fungi and protozoa. Intriguingly, TLRs bind their "non-self" cognate ligand without known maturation or selection. One set of TLR ligands, the Outer Membrane Proteins (OMPs), or porins, are transmembrane 2-barrel proteins. The method of universal recognition of a group of proteins that have large variability in their physical properties is difficult to envision. We hypothesize that TLRs initially scan outer membrane proteins based on electrostatic attraction. We further hypothesize that once TLRs are attracted to a membrane protein, they bind to main chain structural elements thus differentiating 2-strands from 1-helices. The goal of this 2-year proposal is to identify how electrostatics contribute to the recognition of OMPs by TLRs. Specifically, we plan to: 1. Identify the structure of the TLR2-PorB complex. We have already determined the structure of PorB by x-ray crystallography, such that both PorB and TLR2 now have available high-resolution structures. We have further co-purified the complex and taken initial electron microscopy imagers to show feasibility of determination of a co-structure. 2. Investigate the contributions of electrostatics to the affinity of the TLR2-PorB complex. We will use salt and chemical disruption to identify if charge-only effects contribute to the affinity of the TLR2-complex. Specifically, we will identify how methylation and acetylation of lysine side chains affects complex affinity. 3. Identify additional combinations of innate immunity receptors that bind OMPs in vitro. While TLR2 and PorB form one signaling complex, recognition of OMPs by other combinations of TLRs may result in different physiological responses. We have cloned 5 innate immunity receptors and 3 OMPs to identify which combinations of receptors and porins are capable of forming a complex. PUBLIC HEALTH RELEVANCE: We are working to define the mechanisms of recognition between toll-like receptors and outer membrane proteins using a structural approach. We use a hybrid of electron microscopy, NMR, and crystallography to investigate this recognition complex, which spans two membranes in vivo.

描述（由申请人提供）：在先天免疫系统中，Toll样受体（TLR）提供抵抗入侵细菌、病毒、真菌和原生动物的前线防御。有趣的是，TLR结合它们的“非自身”同源配体而不需要已知的成熟或选择。一组TLR配体，外膜蛋白（OMP）或孔蛋白，是跨膜2-桶蛋白。普遍识别一组蛋白质的方法在其物理性质上具有很大的变化性，这是很难想象的。我们假设TLR最初基于静电吸引扫描外膜蛋白。我们进一步假设，一旦TLR被吸引到膜蛋白，它们结合到主链结构元件，从而区分2-链和1-螺旋。本2年提案的目标是确定静电如何有助于TLR识别OMP。具体而言，我们计划：1。确定TLR 2-PorB复合物的结构。我们已经通过X射线晶体学确定了PorB的结构，因此PorB和TLR 2现在都具有可用的高分辨率结构。我们已经进一步共纯化的复合物，并采取了初始的电子显微镜成像显示的可行性，确定一个共同的结构。2.研究静电对TLR 2-PorB复合物亲和力的影响。我们将使用盐和化学破坏，以确定是否仅电荷效应有助于TLR 2复合物的亲和力。具体来说，我们将确定赖氨酸侧链的甲基化和乙酰化如何影响复合物亲和力。3.鉴定在体外结合OMP的先天免疫受体的其他组合。虽然TLR 2和PorB形成一个信号传导复合物，但其他TLRs组合对OMP的识别可能导致不同的生理反应。我们已经克隆了5种先天免疫受体和3种外膜蛋白，以鉴定哪些受体和孔蛋白的组合能够形成复合物。 公共卫生相关性：我们正在努力使用结构方法定义Toll样受体和外膜蛋白之间的识别机制。我们使用电子显微镜，核磁共振和晶体学的混合来研究这种识别复合物，它在体内跨越两个膜。