Structural Characterization of 4-1BB-Galectin-9 Complexes

描述(申请人提供)：肿瘤坏死因子受体超家族中的蛋白质(TNFSF/TNFRSF)在免疫系统的细胞上表达，也在非淋巴结构细胞上表达，如基质细胞、上皮细胞和内皮细胞。它们在调节许多器官的细胞间通讯方面有广泛的活动。肿瘤坏死因子家族分子具有典型的具有夹心胶卷结构的肿瘤坏死因子同源结构域，而TNFR家族分子含有数量可变的与配体结合的胞外富含半胱氨酸的结构域。TNFSF主要以细胞表面或可溶性同源三聚体的形式发挥活性，迄今已解决的11个相互作用的结晶络合物表明，三聚体与3个单独的TNFRSF单体结合。目前尚不清楚单个三聚体复合体是否足以激活细胞，但可能还需要形成基本三聚体的寡聚体来最大限度地传递信号。由于配体结构的不同以及受体识别它们的方式的不同，每个TNFRSF如何形成一个功能信号单位可能会有所不同。TNFSF三聚体分为三类：常规/钟状、EF-二硫键连接/球状和发散/开花状。这些结构被认为是当TNFRSF单体的配体结合到细胞膜上时调节它们之间的间距，这已经被理论上控制连接到信号转导通路的适配器在细胞内的募集。我们研究了最近结晶的4-1BBL(CD137L/TNFSF9)，它是发散/盛开的花状TNFSF分子中最开放的结构。这表明它可能不能像其他TNFSF蛋白那样有效地聚集3个单体4-1BB(CD137/TNFRSF9)分子，或者可能不能有效地允许低聚物形成。因此，4-1BBL可能需要额外的共受体蛋白来参与4-1BB，从而聚集成一个功能齐全的信号单位。我们现在已经发现，4-1BB与Galectin-9(Galectin-9)相互作用，Galectin-9(Galectin-9)是动物凝集素家族的成员，它部分通过碳水化合物与蛋白质相互作用。在免疫细胞的功能研究和体内疾病模型中，我们发现Gal-9是4-1BB活性所必需的。我们假设Gal-9是4-1BB的共同受体蛋白，使4-1BB单体能够复合成一个功能信号单位。我们将用X射线结晶学方法测定人4-1BB与Gal-9的络合物和4-1BB与4-1BBL的络合物的三维结构，并尝试结晶这三个分子的三元配合物。这些研究将为TNFR家族中的结构相互作用定义一个新的范式。

DESCRIPTION (provided by applicant): Proteins in the tumor necrosis factor receptor superfamilies' (TNFSF/TNFRSF) are expressed on cells of the immune system, as well as on non-lymphoid structural cells such as stromal, epithelial, and endothelial cells. They have a broad range of activities in regulating cell-cell communication in many organs. TNF family molecules have canonical TNF homology domains with ¿-sandwich jelly-roll structures, and TNFR family molecules contain variable numbers of ligand-binding extracellular cysteine-rich domains (CRDs). TNFSF are active largely as cell-surface or soluble homotrimers, and the crystallized complexes of 11 interactions solved to date show that the trimers engage 3 individual TNFRSF monomers. Whether a single trimeric complex is sufficient for cellular activation is not known, but oligomers of the basic trimer may also need to form for maximal signaling. How each TNFRSF forms a functional signaling unit likely differs due to variations in the ligand structures as well as how they are recognized by the receptors. TNFSF trimers have been subdivided into 3 groups: conventional/bell-shaped, EF-disulfide bond linked/globular, and divergent/blooming flower-like. These structures have been suggested to regulate the spacing between TNFRSF monomers when binding their ligands on the cell membrane which has been theorized to control the intracellular recruitment of adaptors that link to signal transduction pathways. We have studied 4-1BBL (CD137L/TNFSF9), which was recently crystallized and shown to exert the most open structure of any of the divergent/blooming flower-like TNFSF molecules. This suggests it may not be able to cluster 3 monomeric 4-1BB (CD137/TNFRSF9) molecules as effectively as other TNFSF proteins, or may not efficiently allow oligomers to form. Therefore 4-1BBL might need additional co-receptor proteins to engage 4-1BB to aggregate into a fully functional signaling unit. We have now discovered that 4-1BB interacts with Galectin-9 (Gal-9), a member of a family of animal lectins that interact with proteins in part via carbohydrates. In functional studies of immune cells, and in disease models in vivo, we have found that Gal-9 is required for the activity of 4-1BB. We hypothesize that Gal-9 is a co-receptor protein for 4-1BB that enables 4-1BB monomers to complex into a functional signaling unit. We will determine the three dimensional structures of human 4-1BB complexed with Gal-9, and of 4-1BB complexed with 4-1BBL, using X-ray crystallography, and also attempt to crystallize a ternary complex of all three molecules. These studies will define a new paradigm for structural interaction in the TNFR family.