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: 7790153
• 负责人: T M Iverson
• 金额: $ 23万
• 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/7790153
• 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; Hemophilus; Homo; Hybrids; Immune response; Immune system; In Vitro; Inflammatory Response; Invaded; Lead; Ligands; Literature; Location; Lysine; Maps; Membrane; Membrane Proteins; Menin; Meningitis; Methods; Methylation; Modification; Molecular; Natural Immunity; Neisseria; 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) 提供针对入侵细菌、病毒、真菌和原生动物的前线防御。有趣的是，TLR 在没有已知的成熟或选择的情况下结合其“非自身”同源配体。一组 TLR 配体、外膜蛋白 (OMP) 或孔蛋白是跨膜 2 桶蛋白。很难想象普遍识别一组物理性质变化较大的蛋白质的方法。我们假设 TLR 最初基于静电吸引力扫描外膜蛋白。我们进一步假设，一旦 TLR 被膜蛋白吸引，它们就会与主链结构元件结合，从而区分 2 链和 1 螺旋。这个为期 2 年的提案的目标是确定静电如何有助于 TLR 识别 OMP。具体来说，我们计划： 1. 鉴定 TLR2-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TLR2现在都具有可用的高分辨率结构。我们进一步共纯化了该复合物，并采用了初始电子显微镜成像仪来显示确定共结构的可行性。 2. 研究静电对 TLR2-PorB 复合物亲和力的贡献。我们将使用盐和化学破坏来确定纯电荷效应是否有助于 TLR2 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杂的亲和力。 3. 鉴定在体外结合 OMP 的其他先天免疫受体组合。虽然 TLR2 和 PorB 形成一种信号复合物，但其他 TLR 组合对 OMP 的识别可能会导致不同的生理反应。我们克隆了 5 个先天免疫受体和 3 个 OMP，以确定哪些受体和孔蛋白的组合能够形成复合物。 公共健康相关性：我们正在努力使用结构方法来定义 Toll 样受体和外膜蛋白之间的识别机制。我们综合使用电子显微镜、核磁共振和晶体学来研究这种在体内跨越两个膜的识别复合物。