Structural characterization of γδ-TCR-ligand interaction

尽管人们对γδ T细胞及其在免疫应答中的重要作用越来越感兴趣，但其机制、γδ TCRs如何与其同源抗原相互作用仍知之甚少。造成这种认识上的差距的主要原因之一是鉴定出的γδ TCR /配体对数量有限，从而导致γδ TCR与相应配体配合物的可用结构数量较少。在这个项目中，我们将使用特定的v - γ 3v δ1+ TCR / HLA-DR （MHC II类）对，通过x射线晶体学从结构上表征γδ TCR与其同源配体之间的分子相互作用，该对是Prinz小组在FOR 2799之前的资助期间鉴定的。为此，我们将重组表达来自三种TCR变体的大量可溶性外结构域，这些变体有望结合相同的配体，但具有不同的结合亲和力。我们也将在昆虫细胞中表达大量的可溶性HLA-DR外畴。由于HLA作为MHC II类分子需要与肽相互作用才能折叠和稳定，因此我们将制作后者作为融合蛋白，并与各种肽一起呈现。我们将从重组蛋白组装复合物，结晶这些复合物，用x射线晶体学确定结构，从而确定这种相互作用的主要决定因素。HLA-DR与多肽融合的表达也将使我们能够利用各种HLA-DR /多肽组合在结构上表征肽的作用。这种结构分析将通过对这种相互作用进行彻底的生化分析来补充，这将提供TCR变体与许多HLA /肽组合的详细动力学结合参数。总之，本项目旨在大大提高对γδ TCRs与其同源抗原相互作用的理解。DFG研究单位2799“通过γδ T细胞受体接收和翻译信号”的科学环境将为可持续交流提供最佳条件，我们将与联盟的所有成员分享我们互补的生化和结构生物学专业知识，以潜在地识别更多的γδ TCR /配体对。

The mechanisms, how γδ TCRs interact with their cognate antigen remain poorly understood in spite of the rising interest in γδ T cells and their crucial role in the immune response. One of the main reasons for this gap in understanding is the limited number of identified γδ TCR / ligand pairs and the resulting small number of available structures of γδ TCRs in complex with their respective ligand.In this project, we will structurally characterize the molecular interaction between γδ TCRs and their cognate ligand by X-ray crystallography using a specific Vγ3Vδ1+ TCR / HLA-DR (MHC class II) pair that was identified by the Prinz group in the previous funding period of FOR 2799. To this end, we will recombinantly express important amounts of soluble ectodomains from three TCR variants that are expected to bind the same ligand but with different binding affinity. We will also express important amounts of a soluble HLA-DR ectodomain in insect cells. Since HLA as MHC class II molecule requires interaction with a peptide for folding and stabilization, we will produce the latter as a fusion protein with various peptides to be presented. We will assemble complexes from the recombinant proteins, crystallize these complexes, determine the structure using X-ray crystallography and thereby identify the main determinants of this interaction. The expression of the HLA-DR in fusion with peptides will also enable us to structurally characterize the role of the peptide using various HLA-DR / peptide combinations. This structural analysis will be complemented by a thorough biochemical analysis of this interaction that will provide detailed kinetic binding parameters of the TCR variants to a number of HLA / peptide combinations.In summary, this project aims to considerably advance the understanding of how γδ TCRs interact with their cognate antigen. The scientific environment of the DFG Research Unit FOR 2799 „Receiving and Translating Signals via the γδ T Cell Receptor“ will provide optimal conditions for a sustainable exchange and we will share our complementary biochemical and structural biology expertise with all members of the consortium to potentially identify additional γδ TCR / ligand pairs.