Structure and Activation of a Multiprotein Signaling Complex

项目摘要 细胞对其微环境的反应能力是由多蛋白信号复合物介导的。一 一种特别重要的多蛋白信号复合物涉及传递信号的细胞表面受体 在结合其他细胞上的蛋白质配体时。这种受体的首要实例是T细胞受体（TCR）。 CD 3复合物，免疫系统的主要受体，对微生物的保护性反应至关重要 和癌症。TCR-CD 3复合物由不同的TCRαβ异源二聚体组成， 具有不变的CD 3二聚体CD 3 εγ、CD 3 εδ和CD 3 ε γ。TCR介导肽-MHC（pMHC） 识别，而CD 3将激活信号转导至T细胞。然而，TCR的机制 pMHC参与启动信号传导仍然是一个谜。我们的目标是了解TCR的早期事件 通过获得以下信息来进行信号传导：1）TCR-CD 3复合物的结构;和2）可能的变构 在结合pMHC后TCR动力学的变化，其被传递至CD 3。这些研究将在 通过一个高度集成的项目，结合了多种方法，技术和调查人员。我们将 研究MHC I类限制性TCR（A6）和MHC II类限制性TCR（MS 2 - 3C 8）。A6识别 一种来自HTLV-1的病毒肽，与HLA-A2结合; MS 2 - 3C 8识别来自髓鞘碱性蛋白的自身肽 与HLA-DR 4结合。我们的目标是：1。野生型TCR的表位定位和基于NMR的对接- CD 3复合物。尽管CD 3的胞外区与TCR的胞外区相互作用，但TCR-1的结晶化是不可能的。 CD 3复合物已被TCR-CD 3相互作用的低亲和力所阻碍。我们将使用核磁共振化学 移位扰动和PRE以确定CD 3和TCR之间的结合表位。这些数据将用于 确定野生型TCR-CD 3复合物的结构。2.亲和力成熟的TCR的结构分析- 通过X射线晶体学测定CD 3复合物。我们将尝试克服TCR与TcR之间的弱关联， 通过使用酵母展示的体外定向进化来稳定TCR-CD 3复合物， 晶化来自NMR的关于结合表位的信息将用于设计TCR突变体文库， 将诱变集中在接触CD 3的区域。3. TCR-CD 3结构的生物学验证。我们 将通过评估结构的影响来验证通过NMR或晶体学鉴定的TCR-CD 3界面， TCR-CD 3界面中的指导突变对复杂组装、信号传导和T细胞发育的影响， 细胞测定和表达突变的TCR和CD 3组分的逆转录小鼠。4.动力学分析 TCRαβ胞外域的游离和pMHC结合状态。我们将讨论变构变化假说， 对两种TCR A6进行游离形式和与pMHC结合的TCR胞外域的溶液NMR分析 和MS 2 - 3C 8。我们将通过MD模拟来解决脂质双层对TCR动力学的作用。我们 结合NMR和MD结果将产生通过TCR-CD 3复合物的信号传导的生物物理模型 其基本特征应该适用于通过其他多蛋白复合物和受体的信号传导。

Project Summary The ability of cells to respond to their microenvironment is mediated by multiprotein signaling complexes. A particularly important type of multiprotein signaling complex involves cell surface receptors that transmit signals upon binding protein ligands on other cells. A premier example of such a receptor is the T cell receptor (TCR)– CD3 complex, a cardinal receptor of the immune system that is essential for protective responses to microbes and cancers. The 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 CD3 transduces activation signals to the T cell. However, 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 architecture of the TCR–CD3 complex; and 2) possible allosteric changes in TCR dynamics upon binding pMHC that are relayed to CD3. These studies will be undertaken through a highly integrated project that combines multiple approaches, technologies, and investigators. We will study both an MHC class I-restricted TCR (A6) and an MHC class II-restricted TCR (MS2-3C8). A6 recognizes a viral peptide from HTLV-1 bound to HLA-A2; MS2-3C8 recognizes a self-peptide from myelin basic protein bound to HLA-DR4. Our Aims are: 1. Epitope mapping and NMR-based docking of the wild-type TCR– CD3 complex. Although the extracellular regions of CD3 interact with those of TCR, crystallization of TCR– CD3 complexes has been thwarted by the low affinity of TCR–CD3 interactions. 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. 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 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 regions that contact CD3. 3. Biological validation of the TCR–CD3 structure. We will validate TCR–CD3 interfaces identified by NMR or crystallography by evaluating the effects of structure- guided mutations in TCR–CD3 interfaces on complex assembly, signaling, and T cell development using cellular assays and retrogenic mice expressing mutated TCR and CD3 components. 4. 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 TCR ectodomains in free form and bound to pMHC, for both TCRs A6 and MS2-3C8. We will address the role of the lipid bilayer on TCR dynamics through MD simulations. Our combined NMR and MD results will generate a biophysical model of signaling through the TCR–CD3 complex whose basic features should apply to signaling through other multiprotein complexes and receptors.