Structural Studies of the MIC/NKG 2D Interaction

描述(申请人提供)：自然杀伤(NK)细胞最近发挥作用 公认的在先天免疫中的关键作用，通过阵列发挥作用 介导识别的刺激性和抑制性细胞表面受体 和信号事件。主要组织相容性复合体(MHC)I类 同系物MIC-A是一种应激诱导的自身抗原，广泛被 NK细胞、CD8+α-βT细胞和携带V51的γ/βT细胞亚群 细胞不依赖于β2-微球蛋白和结合肽。因此， MIC-A已被提议“在监测中发挥重要作用 由先天免疫系统转化、感染和损伤的细胞 肠上皮和上皮源性肿瘤。麦克风-A表彰 这些细胞是通过与刺激性NK受体相互作用来调节的。 NKG2D是C型凝集素样蛋白家族的一个分歧成员，它是一种 NK细胞受体NKG2家族其他成员的远亲 CD94。我们最近对三维结构的结晶学分析 揭示了一个戏剧性改变的MHC I类折叠，包括细节和 全局域组织。我们建议将生物化学和生物化学 NK细胞受体NKG2D和NKG2D相互作用的结构细节 MIC蛋白质家族。这一知识将使我们能够理解 重要的NK识别事件，由NKG2D介导，其目标显然是 与α-βT细胞受体或其他NK细胞受体不同， 识别更多传统的MHC I类蛋白和同源物。我们将实现 这一目标是通过1)表达可溶形式的胞外区 MIC蛋白家族和NKG2D的一系列成员，使用多个 表达系统；2)通过定性和定量确定结合亲和力 定量分析；3)通过以下方式描述MIC-A上建议的相互作用部位 定点突变分析；以及4)结晶和测定 单个蛋白质及其合适的复合体的三维结构 通过X射线结晶学。我们还将确定糖基化的影响 对i)MIC/NKG2D相互作用；ii)这些蛋白质的稳定性；以及 三)它们的结构。自本申请书首次递交以来，我们 明确了我们计划研究的三个物种，包括 MIC-AfNKG2D复合体，并收集了一个SPR结合的初步数据 相互作用(杆状病毒表达的MIC-A和细菌表达的NKG2D)。

DESCRIPTION (provided by applicant): Natural killer (NK) cells play a recently recognized and critical role in innate immunity, functioning through an array of stimulatory and inhibitory cell-surface receptors that mediate recognition and signaling events. The major histocompatibility complex (MHC) class I homolog MIC-A is a stress-inducible self antigen that is broadly recognized by NK cells, CD8+ alpha beta T cells and the V51-bearing subset of gamma/delta T cells independent of beta2-microglobulin and bound peptides. Consequently, MIC-A has been proposed to "play an important role in the surveillance of transformed, infected and damaged cells" by the innate immune system in intestinal epithelium and epithelially derived tumors. MIC-A recognition by these cells is mediated through interactions with the stimulatory NK receptor NKG2D, a divergent member of the C-type lectin-like protein family and a distant relative of other members of the NKG2 family of NK cell receptors and CD94. Our recent crystallographic analysis of the three-dimensional structure of MIC-A reveals a dramatically altered MHC class I fold, both in detail and overall domain organization. We propose to characterize the biochemical and structural details of the interaction between the NK cell receptor NKG2D and the MIC family of proteins. This knowledge will allow us to understand an important NK recognition event, mediated by NKG2D, whose target is clearly unlike that of alpha beta T cell receptors or other NK cell receptors which recognize more conventional MHC class I proteins and homologs. We will achieve this goal by 1) expressing soluble forms of the extracellular domains of a series of members of the MIC protein family and NKG2D, using multiple expression systems; 2) determining binding affinities by both qualitative and quantitative assays; 3) delineating a proposed interaction site on MIC-A by site-directed mutational analysis; and 4) crystallizing and determining the three-dimensional structures of individual proteins and appropriate complexes by x-ray crystallography. We will also 5) determine the effect glycosylation has on i) the MIC/NKG2D interaction; ii) the stability of these proteins; and iii) their structures. Since the initial submission of this application, we have crystallized three of the species we propose to study, including the MIC-AfNKG2D complex, and have collected preliminary SPR binding data for one interaction (baculovirus-expressed MIC-A and bacterially-expressed NKG2D).