Structure and Function of Toll-like Receptors

脊椎动物对感染的免疫反应始于天然免疫系统对病原体保守的分子特征的识别，称为PAMPs(病原体相关分子模式)，引发立即且往往是大规模的炎症反应。与生俱来的反应抑制了病原体，但也在后天免疫的产生中发挥了关键作用。先天系统对PAMPs的识别是由多种受体介导的，其中Toll样受体(TLRs)起着重要的作用。与获得性免疫的抗原受体不同，TLRs由有限数量的生殖系基因编码，在人类中为10个；然而，尽管数量很少，TLRs识别的PAMP种类非常广泛，包括糖脂、蛋白质和核酸。TLRs识别PAMP的分子基础是我的实验室的主要兴趣所在。我们与David Davies博士(LMB，NIDDK)合作，表达了TLR3胞外区(ECD)的镁含量，并通过X射线结晶学确定了其结构。双链(DS)RNA是许多病毒的分子特征，它结合并激活TLR3。TLR3-ECD的结构由一个23圈的螺线管组成，弯曲成马蹄形，凹面上有一个大的贝塔板。该分子是高度糖基化的，除了马蹄铁的一个侧面完全不含糖，根据突变分析，这一面被预测为配体结合的位置。为了确定TLR3与其配体的结合方式，我们首先研究了dsRNA寡核苷酸与TLR3-ECD蛋白在溶液中的相互作用。我们发现，纯化的TLR3-ECD通过定义的配体结合位点与dsRNA特异性结合，亲和力随着缓冲液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。这些二聚体通过成对的两个TLR3-ECD之间的协同作用来稳定，多个TLR3-ECD二聚体与长dsRNA结合。与TLR3-ECD(40-50bp)形成稳定络合物的最小寡核苷酸也是在细胞中激活TLR3的最小dsRNA。因此，我们证明了最小的TLR3信号单位是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、结晶并求解了TLR3信号复合体的结构，该复合体由两个TLR3-ECD分子与一个46bp的dsRNA寡核苷酸结合组成。该复合体中的两个TLR3-ECD相互面对，并通过dsRNA分子两侧的双重对称联系在一起。我们确定了稳定该复合体的三个分子间接触。DsRNA与每个mTLR3-ECD的无糖表面上的N-末端和C-末端结合。C-末端相互垂直，而N-末端在线形dsRNA分子的相对两端延伸。该复合体的长度为141，与形成复合体所需的最小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-末端的封闭区相互结合，这是形成二聚体的原因。MTLR3-ECD的整体结构在与dsRNA结合后不会改变，支持一种信号机制，即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反应。该复合体中的dsRNA保持了典型的A-DNA样结构，其中核糖-磷酸骨架和沟槽的位置是结合的主要决定因素。MTLR3-ECD与糖-磷酸骨架相互作用，但不与单个碱基相互作用。这解释了为什么TLR3对任何特定的核苷酸序列缺乏特异性。任何一个结合位点的点突变使TLR3功能丧失，表明了一种三个低亲和力位点协同作用形成稳定的信号复合体的机制。

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.