Structural Studies of the MIC/NKG 2D Interaction

描述（由申请人提供）：自然杀伤（NK）细胞最近发挥了 在先天免疫中的公认和关键作用，通过一系列 刺激性和抑制性细胞表面受体介导的识别 和信号事件。主要组织相容性复合体（MHC）I类 同源物MIC-A是一种应激诱导的自身抗原， NK细胞、CD 8 + α β T细胞和携带V51的γ/δ T细胞亚群 不依赖于β 2-微球蛋白和结合肽的细胞。因此，委员会认为， 已提议中等收入国家-阿富汗“在监测 转化，感染和受损的细胞”的先天免疫系统， 肠上皮和上皮来源的肿瘤。MIC-A认证 这些细胞通过与刺激性NK受体的相互作用来介导 NKG 2D是C型凝集素样蛋白家族的一个分歧成员， NK细胞受体NKG 2家族其他成员的远亲， CD94。我们最近的三维结构的晶体学分析 的MIC-A揭示了一个显着改变的MHC I类折叠，无论是在细节上， 整个域组织。我们建议将生物化学和 NK细胞受体NKG 2D与 MIC蛋白质家族。这些知识将使我们能够理解一个 重要的NK识别事件，由NKG 2D介导，其靶点明确 与α β T细胞受体或其它NK细胞受体不同， 识别更常规的MHC I类蛋白和同源物。我们将实现 1）表达可溶形式的细胞外结构域， MIC蛋白家族和NKG 2D的一系列成员，使用多个 表达系统; 2）通过定性和 定量测定; 3）通过以下方法描绘MIC-A上的拟议相互作用位点： 定点突变分析;和4）结晶和确定 单个蛋白质和适当复合物的三维结构 通过X射线晶体学。我们还将确定糖基化对 具有i）MIC/NKG 2D相互作用; ii）这些蛋白质的稳定性;和 （三）结构。自首次提交本申请以来，我们 已经结晶了我们打算研究的三个物种，包括 MIC-AfNKG 2D复合物，并收集了一种药物的初步SPR结合数据。 相互作用（杆状病毒表达的MIC-A和细菌表达的NKG 2D）。

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).