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

描述（由申请人提供）：肿瘤坏死因子受体超家族（TNFSF/TNFRSF）中的蛋白在免疫系统细胞以及非淋巴样结构细胞（如基质细胞、上皮细胞和内皮细胞）上表达。在许多器官中，它们在调节细胞间通讯方面具有广泛的活性。TNF家族分子具有典型的具有三明治果冻卷结构的TNF同源结构域，并且TNFR家族分子含有可变数量的配体结合的细胞外富含半胱氨酸的结构域（CRDs）。TNFRSF主要以细胞表面或可溶性三聚体的形式活跃，迄今为止已解决的11种相互作用的结晶配合物表明，三聚体与3个单独的TNFRSF单体结合。单个三聚体复合物是否足以激活细胞尚不清楚，但基本三聚体的低聚物也可能需要形成最大的信号传导。由于配体结构的变化以及受体对它们的识别方式不同，每个TNFRSF如何形成一个功能性信号单元可能会有所不同。TNFSF三聚体被细分为3类：常规/钟形，ef -二硫键连接/球形和发散/开花状。这些结构被认为可以调节TNFRSF单体在细胞膜上结合配体时的间距，理论上可以控制连接信号转导途径的接头在细胞内的募集。我们研究了4-1BBL (CD137L/TNFSF9)，它是最近结晶的，并显示出在所有分化/开花的TNFSF分子中最开放的结构。这表明它可能无法像其他TNFSF蛋白那样有效地聚集3个单体4-1BB （CD137/TNFRSF9）分子，或者可能无法有效地形成低聚物。因此，4-1BBL可能需要额外的共受体蛋白来参与4-1BB聚集成一个功能齐全的信号单元。我们现在已经发现4-1BB与半乳糖凝集素-9 （Gal-9）相互作用，半乳糖凝集素是动物凝集素家族的一员，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的共受体蛋白，使4-1BB单体复合体成为功能信号单元。我们将利用x射线晶体学确定人体4-1BB与Gal-9络合的三维结构，以及4-1BB与4-1BBL络合的三维结构，并尝试结晶这三种分子的三元配合物。这些研究将定义TNFR家族结构相互作用的新范式。

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.