Structure and Function of Toll-like Receptors

Toll样受体的结构和功能

• 批准号: 7732935
• 负责人: David M. Segal
• 金额: $ 65.89万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732935
• 关键词: Accounting; Adjuvant; Affinity; Anti-Inflammatory Agents; Antigen Receptors; Base Sequence; Binding; Binding Sites; Buffers; C-terminal; Cells; Collaborations; Complex; Decompression Sickness; Dimerization; Double-Stranded RNA; Epitopes; Extracellular Domain; Face; Generations; Genes; Glycolipids; Goals; Human; Immune response; Immune system; Individual; Infection; Inflammatory Response; Laboratories; Lateral; Length; Ligand Binding; Ligands; Location; Major Groove; Mediating; Molecular; Molecular Profiling; N-terminal; National Institute of Diabetes and Digestive and Kidney Diseases; Nucleic Acids; Numbers; Oligonucleotides; Pattern; Play; Point Mutation; Polysaccharides; Positioning Attribute; Proteins; Purpose; Role; Shapes; Side; Signal Transduction; Site; Solutions; Specificity; Structure; Sugar Phosphates; Surface; System; TLR3 gene; Toll-like receptors; Vaccines; Vertebral column; Virus; X-Ray Crystallography; acquired immunity; base; beta pleated sheet; design; dimer; human wyatt protein; improved; interest; mouse wyatt protein; pathogen; receptor; response; ribose phosphate; size

The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, of which the Toll-like Receptors (TLRs) play a prominent role. Unlike the antigen receptors of acquired immunity, the TLRs are encoded by a limited number of germline genes, ten in humans; however, in spite of their small numbers, the TLRs recognize a remarkably wide variety of PAMPs including glycolipids, proteins, and nucleic acids. The molecular basis for the recognition of PAMPs by TLRs is a main interest of my laboratory. In collaboration with Dr. David Davies (LMB, NIDDK), we have expressed mg amounts of the extracellular domain (ECD) of TLR3, and have determined its structure by X-ray crystallography. Double-stranded (ds) RNA, a molecular signature of many viruses, binds and activates TLR3. The structure of TLR3-ECD consists of a solenoid of 23 turns, bent into a horseshoe shape, with a large beta-sheet on the concave surface. The molecule is heavily glycosylated, except that one lateral face of the horseshoe is totally devoid of glycan, and this face was predicted, based on mutational analyses, to be the location for ligand binding. To determine how TLR3 binds its ligand, we first studied the interaction of dsRNA oligos with TLR3-ECD protein in solution. We found that purified TLR3-ECD binds dsRNA specifically via a defined ligand-binding site, with an affinity that increases with buffer acidification and ligand size. TLR3-ECD is monomeric in solution, but it forms dimers when bound to dsRNA. These dimers are stabilized by cooperative interactions between the two TLR3-ECDs in a pair, and multiple TLR3-ECD dimers bind to long dsRNAs. The smallest oligonucleotides that form stable complexes with TLR3-ECD (40-50 bp) are also the smallest dsRNAs that activate TLR3 in cells. Thus, we demonstrated that the minimal TLR3 signaling unit is a ligand-bound dimer of TLR3 molecules. To determine the molecular basis for ligand binding and signaling, we isolated, crystallized, and solved the structure of the TLR3 signaling complex, consisting of two TLR3-ECD molecules bound to one 46 bp dsRNA oligonucleotide. The two TLR3-ECDs in the complex face one another, and are related by two-fold symmetry on opposite sides of the dsRNA molecule. We identified three intermolecular contacts that stabilize the complex. The dsRNA interacts with both an N-terminal and a C-terminal site on the glycan-free surface of each mTLR3-ECD. The C-terminal sites are directly across from each other while the N-terminal sites are outstretched at opposing ends of the linear dsRNA molecule. The length of the complex is 141 , which corresponds well with the minimal dsRNA oligo size required for complex formation. In addition, the two TLR3-ECD molecules of the dimer bind each other at the C-terminal capping domain, which accounts for dimer formation. The overall structure of mTLR3-ECD does not change upon binding to dsRNA, supporting a signaling mechanism in which ligand-induced receptor dimerization brings the two cytoplasmic TIR domains into contact, thus triggering a downstream signaling cascade. The dsRNA in the complex retains a typical A-DNA-like structure, in which the ribose-phosphate backbone and the position of the grooves are the major determinants of binding. The mTLR3-ECD interacts with the sugar-phosphate backbones, but not with individual bases. This explains why TLR3 lacks specificity for any particular nucleotide sequence. Point mutation in any one of the binding sites abrogate TLR3 function, indicating a mechanism in which three low affinity sites act cooperatively to form a stable signaling complex.

脊椎动物对感染的免疫反应始于先天免疫系统对病原体保守分子特征的识别，称为PAMPs（病原体相关分子模式），引发立即且通常是大量的炎症反应。先天反应控制病原体，但在获得性免疫的产生中也起着至关重要的作用。先天系统对PAMPs的识别是由许多受体介导的，其中toll样受体（TLRs）起着突出的作用。与获得性免疫的抗原受体不同，tlr由有限数量的种系基因编码，在人类中有10个；然而，尽管它们的数量很少，但tlr识别的pamp种类非常广泛，包括糖脂、蛋白质和核酸。tlr识别PAMPs的分子基础是我实验室的主要兴趣。我们与David Davies博士（LMB， NIDDK）合作，表达了TLR3细胞外结构域（ECD）的mg量，并通过x射线晶体学确定了其结构。双链RNA （ds）是许多病毒的分子特征，结合并激活TLR3。TLR3-ECD的结构包括一个23圈的电磁阀，弯曲成马蹄形，凹表面有一个大的β片。这个分子是重度糖基化的，除了马蹄形的一个侧面完全没有糖基化，根据突变分析，这面被预测为配体结合的位置。为了确定TLR3如何与其配体结合，我们首先研究了溶液中dsRNA寡核苷酸与TLR3- ecd蛋白的相互作用。我们发现纯化的TLR3-ECD通过一个确定的配体结合位点特异性地结合dsRNA，其亲和力随着缓冲酸化和配体大小的增加而增加。TLR3-ECD在溶液中为单体，但与dsRNA结合时形成二聚体。这些二聚体通过一对TLR3-ECD之间的合作相互作用而稳定，并且多个TLR3-ECD二聚体结合到长dsrna上。与TLR3- ecd形成稳定复合物的最小寡核苷酸（40-50 bp）也是细胞中激活TLR3的最小dsRNAs。因此，我们证明了最小的TLR3信号单元是TLR3分子的配体结合二聚体。为了确定配体结合和信号转导的分子基础，我们分离、结晶并解析了TLR3信号转导复合物的结构，该复合物由两个TLR3- ecd分子结合一个46 bp的dsRNA寡核苷酸组成。复合物中的两个tlr3 - ecd彼此面对，并通过dsRNA分子两侧的双重对称联系在一起。我们确定了三个稳定复合物的分子间接触。dsRNA与每个mTLR3-ECD无聚糖表面的n端和c端位点相互作用。c端位点彼此直接相连，而n端位点则在线性dsRNA分子的两端伸出。该络合物的长度为141，这与形成络合物所需的最小dsRNA寡核苷酸大小很好地对应。此外，二聚体的两个TLR3-ECD分子在c端旋盖结构域相互结合，这也是二聚体形成的原因。mTLR3-ECD的整体结构在与dsRNA结合后不会发生改变，这支持了一种配体诱导的受体二聚化使两个细胞质TIR结构域接触从而触发下游信号级联的信号机制。复合体中的dsRNA保留了典型的a - dna样结构，其中核糖-磷酸主链和凹槽的位置是结合的主要决定因素。mTLR3-ECD与糖-磷酸主干相互作用，但不与单个碱基相互作用。这就解释了为什么TLR3对任何特定的核苷酸序列都缺乏特异性。任何一个结合位点的点突变都会使TLR3的功能失效，这表明三个低亲和力位点协同作用形成稳定的信号复合物的机制。

项目成果

Induction of dendritic cell maturation by pertussis toxin and its B subunit differentially initiate Toll-like receptor 4-dependent signal transduction pathways.

• DOI: 10.1016/j.exphem.2006.04.025
• 发表时间: 2006-08
• 期刊: Experimental hematology
• 影响因子: 2.6
• 作者: Z. Wang;De Yang;Qian Chen;C. Leifer;D. Segal;S. Su;R. Caspi;Zack Howard;J. Oppenheim