Structural Analysis of the TCR-CD3 Complex and TCR Signaling

 描述(申请人提供)：T细胞受体(Tcr)-CD3复合体由不同的Tcrαβ异二聚体组成，与不变的CD3二聚体CD3εγ、CD3εδ和CD3ζζ非共价结合。TCR介导肽-MHC(PMHC)识别，而CD3分子将激活信号转导到T细胞。尽管人们对下游T细胞信号通路有很多了解，但pMHC参与TCR启动信号传递的机制仍然是一个谜。我们的目标是通过获得以下信息来了解TCR信号的早期事件：1)TCR-CD3复合体的空间组织；以及2)与传递到CD3的pMHC结合时，TCR构象和/或动力学可能的变构变化。这些研究将通过一个高度综合的项目进行，该项目结合了多种方法、技术和调查人员。为了确保我们发现的一般性，我们将研究MHC I类限制性TCR(A6)和MHC II类限制性TCR(MS2-3C8)。A6识别与人类白细胞抗原A2结合的HTLV-1病毒抗原；MS2-3C8识别与人类白细胞抗原DR4结合的髓鞘碱性蛋白的自体肽。我们的具体目标是：1.用核磁共振方法确定野生型TCR-CD3复合体的结构。虽然已知CD3的胞外区与TCR的胞外区相互作用，但所有使TCR-CD3复合体结晶的尝试都因TCR-CD3在溶液中的相互作用亲和力很低而受阻。我们将使用核磁共振、化学位移扰动和PRE来确定CD3和TCR之间的结合表位。这些数据将被用于确定野生型TCR-CD3复合体的结构。我们将讨论pMHC结合是否改变TCR-CD3相互作用，可能触发T细胞信号。2.亲和成熟的TCR-CD3络合物的X射线结晶学结构分析我们将尝试通过体外定向进化来克服TCR和CD3胞外区之间的弱结合，使用酵母表面展示来稳定TCR-CD3复合体的结晶。有关核磁共振结合表位的信息将被用来设计TCR突变库，方法是将突变集中在那些与CD3接触的区域(反之亦然)。3.TCR-CD3结构的生物学验证。我们将通过评估TCR-CD3界面的基于结构的突变对复杂组装、细胞表面表达、信号转导以及T细胞功能和发育的影响，来验证通过核磁共振或X射线结晶学鉴定的TCR-CD3界面。TCR-CD3复合体将使用2A连接的逆转录病毒载体进行重组，以产生T细胞或表达TCR和CD3组分的特定组合的逆转录小鼠。4.TcRαβ胞外区自由态和结合态的结构和动力学分析我们将通过对TCRA6和MS2-3C8的自由形式并与pMHC结合的全长TCR胞外结构域的溶液核磁共振分析来解决变构变化假说。我们将研究pMHC连接是否导致TCR构象和/或动力学的变化，如果是，则将这些变化与TCR触发的可能机制联系起来，涉及与CD3的相互作用。

 DESCRIPTION (provided by applicant): The T cell receptor (TCR)-CD3 complex is composed of a diverse TCRαβ heterodimer noncovalently associated with the invariant CD3 dimers CD3εγ, CD3εδ, and CD3ζζ. The TCR mediates peptide-MHC (pMHC) recognition, while the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by pMHC initiates signaling remains a mystery. Our goal is to understand the early events of TCR signaling by obtaining information on: 1) the spatial organization of the TCR-CD3 complex; and 2) possible allosteric changes in TCR conformation and/or dynamics upon binding pMHC that are relayed to CD3. These studies will be undertaken through a highly integrated and comprehensive project that combines multiple approaches, technologies, and investigators. To assure the generality of our findings, we will study both an MHC class I-restricted TCR (A6) and an MHC class II-restricted TCR (MS2-3C8). A6 recognizes a viral antigen from HTLV-1 bound to HLA-A2; MS2-3C8 recognizes a self-peptide from myelin basic protein bound to HLA-DR4. Our Specific Aims are: 1. Structure determination of the wild-type TCR-CD3 complex by NMR. Although the extracellular regions of CD3 are known to interact with the extracellular regions of the TCR, all attempts to crystallize TCR-CD3 complexes have been thwarted by the very low affinity of TCR-CD3 interactions in solution. We will employ NMR chemical shift perturbation and PRE to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the wild-type TCR-CD3 complex. We will address whether pMHC binding alters TCR-CD3 interactions, possibly triggering T cell signaling. 2. Structural analysis of affinity- matured TCR-CD3 complexes by X-ray crystallography. We will attempt to overcome the weak association between TCR and CD3 ectodomains by in vitro directed evolution using yeast surface display to stabilize TCR-CD3 complexes for crystallization. Information on binding epitopes from NMR will be used to design TCR mutant libraries by focusing mutagenesis on those regions that contact CD3 (or vice versa). 3. Biological validation of the TCR-CD3 structure. We will validate TCR-CD3 interfaces identified by NMR or X-ray crystallography by evaluating the effects of structure-based mutations in TCR-CD3 interfaces on complex assembly, cell surface expression, signaling, and T cell function and development. The TCR-CD3 complex will be reconstituted using 2A-linked retroviral vectors to generate T cells or retrogenic mice expressing defined combinations of TCR and CD3 components. 4. Structural and dynamics analysis of free and pMHC-bound states of TCRαβ ectodomains. We will address the allosteric change hypothesis by carrying out solution NMR analysis of full-length TCR ectodomains in free form and bound to pMHC, for both TCRs A6 and MS2- 3C8. We will investigate whether pMHC ligation induces changes in TCR conformation and/or dynamics and, if so, relate these changes to a possible mechanism for TCR triggering involving interactions with CD3.