TOLL-SPATZLE COMPLEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Toll受体首先在果蝇(一种果蝇)中被鉴定，并在胚胎发育和先天免疫反应中起重要作用。最近在脊椎动物基因组中发现了类似的受体，称为Toll样受体(TLRs)，在先天免疫中发挥着重要作用。TLRs识别并对多种病原体衍生物质做出反应。Toll蛋白和TLRs属于I型跨膜糖蛋白家族，在N-端和C-端有几个富含亮氨酸重复序列(LRR)的富含半胱氨酸的封顶元件。 当其胞外结构域与受体配体结合时，Toll受体(和TLRs)就会被激活。细胞因子Spétzle(106个氨基酸基序，分子质量为24 kDa的二聚体)就是这样一种激活Toll受体的配体。SP？tzle通过二硫键形成二聚体，已知在N-末端与Toll蛋白结合。Toll蛋白与Sp？tzle的相互作用导致两个Toll蛋白的胞外区发生交联，进而导致Toll的信号转导。通过研究配体诱导的Toll蛋白二聚化，可以更好地了解Toll蛋白的信号机制。 尽管人们对Toll受体(分子质量为110 kDa)的结构性质、与SP的相互作用和二聚化过程越来越感兴趣，但现有文献中缺乏结构信息。最近，Toll样受体(TLR3)的结构已被X射线结晶学解析到原子分辨率。我们实验室对从电子显微镜图像中重建的单粒子结构感兴趣，并解决了在人类细胞中表达的TLR3胞外区的结构。最近，我们利用负染色的EM显微图像和EMAN软件解决了Toll蛋白和Toll-SP？tzle复合体的结构，分辨率约为25？Toll和Toll-Sp？tzle结构的叠加清楚地识别了Toll-Sp？tzle复合体中的Sp？tzle，从而识别了蛋白质的N-末端。此外，我们还有形成Toll蛋白二聚体的Toll-Sp？tzle复合体。用负染色的电子显微照片将结构解析到~30°的分辨率。结构分析表明，这两个Toll蛋白通过其C端相互作用，在其N端结合了sp？tzle。有趣的是，还观察到了蛋白质N末端附近的另一种相互作用(靠近Sp？tzle结合部位)。这种二聚体复合体的高分辨率结构将有助于我们获得N-末端相互作用的细节。通过这项初步研究，我们建议对Toll蛋白的二聚体进行低温电子显微镜分析，以进行高分辨率的结构重建。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Toll receptors were first characterized in Drosophila melanogaster (a fruit fly) and are important for embryonic development and innate immune response. Similar receptors called Toll-like receptors (TLRs) were recently found in vertebrate genomes and play a major role in innate immunity. TLRs recognize and respond to a wide variety of pathogen-derived substances. Toll proteins and TLRs belong to a family of type I transmembrane glycoproteins with several blocks of Leucine rich repeat (LRR) regions with cysteine-rich capping elements at N- and C-terminus. Toll receptors (and TLRs) get activated when its ectodomain binds to a receptor ligand. Cytokine Sp¿¿tzle (106 amino acid motif with a molecular mass of 24 kDa in its dimeric form) is one such ligand which activates toll receptors. Sp¿¿tzle forms dimers through disulphide bonds and known to bind to Toll protein at the N-terminus. The interaction of Toll protein with Sp¿¿tzle leads to the crosslinking of ectodomains of two Toll proteins, which then leads to the signal transduction by Toll. The signaling mechanism of Toll proteins can be best understood by studying the ligand induced dimerization of the toll proteins. Despite the growing interest in structural properties of Toll receptors (molecular mass 110 kDa), their interaction with sp¿¿tzle and dimerization process, there is a lack of structural information in the available literature. Recently, the structure of Toll-like receptor (TLR3) has been solved by X-ray crystallography to an atomic resolution. Our lab is interested in studying single-particle structures reconstituted from electron microscopy images and have solved the structure of TLR3 ectodomain expressed in human cells. Recently, we have solved the structure of Toll protein and Toll-sp¿¿tzle complex using negatively stained EM micrographs and EMAN software to a resolution of about 25 ¿ . Superposition of Toll and Toll-Sp¿¿tzle structures clearly identifies the Sp¿¿tzle in the Toll-Sp¿¿tzle complex and hence the N-terminus of the protein. Also, we have a Toll-Sp¿¿tzle complex that formed dimers of toll proteins. The structure has been solved to a resolution of ~ 30 ¿ using negatively stained electron micrographs. The structure shows that the two toll proteins with sp¿¿tzle bound at their N-terminus interact with each other through their C-terminus. Interestingly, another interaction near the N-terminus of the proteins (near Sp¿¿tzle binding site) was also observed. High resolution structure of this dimer complex will help us to get the details of the interaction at the N-terminus. With this preliminary study, we are proposing to perform the cryoEM analysis of the toll protein in its dimeric form for a high resolution structure reconstruction.