Physical basis for T cell receptor binding and activity

描述(由申请人提供)：通过结晶学方法对TCR-肽/MHC复合体的可视化提供了对TCR-肽/MHC相互作用性质的巨大洞察。然而，结构是静态的，不能揭示T细胞受体如何与其配体结合的分子机制。结构并不表明TCR和肽/MHC分子结合和解离的速度，也不表明界面内的细节对复合体稳定性的贡献程度。结构提供的有关蛋白质在形成复合体时的动态运动的信息很少。结合动力学和热力学与T细胞反应的类型和效力相关，环的流动性与交叉反应和配体识别有关。在这里，将对TCR-多肽/MHC相互作用进行详细的研究，以解决结晶学尚未回答的问题。具体地说，将研究多个T细胞受体之间的相互作用，这些受体都识别由HLA-A2呈递的Tax肽。其中两个受体A6和B7与配体形成了几乎完全不同的接触，但这两个受体复合体的整体结构和功能反应非常相似。其他TAX/HLA-A2特异性受体的初步序列数据表明，它们同样与不同的氨基酸结合。这表明，给定的受体可以通过多种方式识别配体。对同一配体的多个受体的研究应能提供对TCRs如何结合肽/MHC的重要洞察力，因为可以进行详细的比较、关联和最终的概括。将进行的实验包括使用滴定量热法、表面等离子体共振和分析超速离心法测定至少七种TCR-TAX/人类白细胞抗原-A2相互作用的结合热力学、动力学和活化能。将进行突变分析，以确定每个界面内的“热点”或“焦点”。这项工作将超越传统的丙氨酸扫描诱变，以确定已确定的热点的物理基础。一个关键的控制将包括利用荧光偏振来确定突变对MHC结合的多肽的影响。还将进行细胞毒性和细胞因子释放的功能测量，允许结合测量与生物活性相关。在已知或模型化的三维结构的背景下解释结合和功能测量将允许结合和活性的决定因素映射到每个界面的结构特征。这项工作的高潮将是试图确定合理操纵TCR结合和活性的一般策略。

DESCRIPTION (provided by applicant): Visualization of TCR-peptide/MHC complexes through crystallographic methods has provided enormous insight into the nature of TCR-peptide/MHC interactions. However, structures are static and do not reveal the molecular mechanisms of how the T cell receptor binds its ligand. Structures do not indicate the rates at which TCRs and peptide/MHC molecules associate and dissociate or the extent to which details within the interface contribute to the stability of the complex. Structures provide little information about the dynamic motions the proteins undergo upon forming the complex. Binding kinetics and thermodynamics have been correlated with the type and potency of T cell response, and loop mobility has been implicated in cross-reactivity and ligand discrimination. Here, a detailed investigation of TCR-peptide/MHC interactions addressing questions left unanswered by crystallography will be performed. Specifically, the interactions between multiple T cell receptors that all recognize the Tax peptide presented by HLA-A2 with will be studied. Two of these receptors, A6 and B7, are known to form almost entirely different contacts with ligand, yet the overall structures and functional responses of the two receptor complexes are extremely similar. Preliminary sequence data for other Tax/HLA-A2 specific receptors suggests that they likewise bind with different amino acids. This indicates that there are multiple ways in which a given receptor can recognize a ligand. Examination of multiple receptors specific for the same ligand should provide significant insight into how TCRs bind peptide/MHC, as detailed comparisons, correlations, and ultimately generalizations can be made. Experiments to be performed include determination of binding thermodynamics, kinetics, and activation energies for at least seven TCR-Tax/HLA-A2 interactions using titration calorimetry, surface plasmon resonance, and analytical ultracentrifugation. Mutational analyses will be performed, allowing determination of "hot spots" or "focal points" within each interface. The work will move beyond traditional alanine scanning mutagenesis in order to determine the physical basis for identified hot spots. A critical control will include determination of the effects mutations have on peptide binding by MHC using fluorescence polarization. Functional measurements of cytotoxicity and cytokine release will also be performed, allowing binding measurements to be correlated with biological activity. Interpretation of binding and functional measurements in the context of either known or modeled three-dimensional structures will permit the determinants of binding and activity to mapped to structural features of each interface. The work will culminate with attempts to identify general strategies for the rational manipulation of TCR binding and activity.