Molecular Interactions Of Lymphoid Cell Receptors

这些项目利用了实验室在研究分子相互作用和分子结构方面的专业知识。对于上述项目(1)，我们开发了一种生成二硫捕获肽/ mhc - 1分子的策略，并表达、折叠、纯化和结晶了其中的几个。特别是，我们已经研究了H2-Dd分子，其中结合间隙中的残基已经突变为半胱氨酸，并在适当的位置用含有半胱氨酸的肽重新折叠这些分子。这些x射线晶体结构证实了二硫捕获肽稳定地固定在肽结合槽中。用截断的肽产生的这些分子的版本表明，这些分子在肽结合槽的一部分是肽稳定的，但在截断的位置是松弛的。结合研究表明，这些分子应该允许绘制与各种细胞和病毒伴侣相互作用的位点。使用一种稍微不同的方法，我们产生了几种具有新型链内二硫键的MHC-I分子，特别是HLA-B*18:01， H2-Dd和H2-Ld。虽然我们最初预计这些物质会表现出更大的热稳定性，但正在进行的研究表明，这种稳定性也与这些MHC分子结合的特定肽有关。进一步的研究不仅要考察其稳定性，而且要考察其作为四聚体染色试剂的生物活性和实用性。对于上述(2)，我们与Berzofsky实验室合作，探索了由小鼠疫苗免疫产生的独特肽，其中组织蛋白酶S位点被操纵以改变交叉呈现。从HIV-MNgp120蛋白中鉴定出三种肽（IGPGRAFYVI、IGPGRAFYV和IGPGRAFYT），它们由H2-Dd分子呈现。我们的实验室检查了这些独特的H2-Dd/肽复合物的x射线结构。三维结构解释了肽的差异结合，也解释了它们各自的四聚体在识别疫苗接种引起的CD8 T细胞群中的行为。For(3)与Shihoko Komine-Aizawa和Mitsuo Honda合作，探讨了CD8 T细胞对编码结核分枝杆菌Ag85B蛋白的重组bcg疫苗免疫小鼠的反应。鉴定了几种h2 - kd限制性Ag85B肽（YYQSGLSIV和YQSGLSIVM），并对它们进行了结晶和x射线结构测定。这些结构表明，变异肽可以以新的构象与H2-Kd等MHC分子结合，并为新疫苗引发的交叉反应性和非交叉反应性T细胞群提供结构见解。肽/H2-Kd四聚体鉴定了Ag85B疫苗诱导的两种不同的T细胞群。特别是，YQSGLSIVM肽以非规范取向结合，其c端残基延伸到肽结合槽之外。在上面列出的第四个子项目中，我们成功地设计了LAG-3蛋白及其纤维蛋白原样蛋白1配体以及各种MHC-II配体。这些新试剂将为研究LAG-3与不同MHC-II构象的相互作用以及评价其与FGL1相互作用的作用提供基础。

These projects take advantage of the laboratory's expertise in studying molecular interactions and molecular structure. For project (1) indicated above, we have developed a strategy for generating disulfide-trapped peptide/MHC-I molecules and have expressed, refolded, purified, and crystallized several of these. In particular, we have examined H2-Dd molecules in which a residue in the binding cleft has been mutated to cysteine and have refolded these with peptides containing cysteine in appropriate positions. X-ray crystallographic structures of these confirm that the disulfide-trapped peptides are stably fixed in the peptide binding groove. Versions of these, produced with truncated peptides, indicate that these molecules are peptide-stabilized in part of the peptide binding groove but are relaxed in the positions of truncation. Binding studies indicate that these molecules should allow mapping of sites of interaction with various cellular and viral chaperones. Using a slightly different approach, we have produced several variations of MHC-I molecules with novel intrachain disulfide bonds, specifically HLA-B*18:01, H2-Dd and H2-Ld. Although we originally expected these to exhibit greater thermal stability, studies underway indicate that the stability is also related to the particular peptide bound by these MHC molecules. Further studies to investigate not only the stability, but the biological activity and utility as tetramer staining reagents are underway. For (2) above, collaborative with the Berzofsky laboratory, we explored unique peptides generated as a result of vaccine immunization of mice in which cathepsin S sites were manipulated to alter cross-presentation. Three peptides (IGPGRAFYVI, IGPGRAFYV, and IGPGRAFYT) from the HIV-MNgp120 protein were identified as presented by the H2-Dd molecule. Our laboratory examined the X-ray structures of these unique H2-Dd/peptide complexes. The three-dimensional structures explain the differential binding of the peptides, and also explain the behavior of their respective tetramers in identifying CD8 T cell populations evoked by vaccination. For (3), carried out collaboratively with Shihoko Komine-Aizawa and Mitsuo Honda, explores the CD8 T cell response to immunization of mice with recombinant BCG-based vaccines that encode the Ag85B protein of M. tuberculosis. Several H2-Kd-restricted Ag85B peptides were identified (YYQSGLSIV and YQSGLSIVM) and we have crystallized and determined the X-ray structures of these. These structures show that variant peptides can bind to MHC molecules like H2-Kd in novel conformations, and offer structural insight into cross-reactive and non-cross-reactive T cell populations primed by the new vaccines. Peptide/H2-Kd tetramers identify two distinct T cell populations elicited by the Ag85B vaccine. In particular, the YQSGLSIVM peptide binds in a non-canonical orientation with its C-terminal residue extending beyond the peptide binding groove. In the fourth sub-project listed above, we have successfully engineered the LAG-3 protein and its fibrinogen like protein 1 ligand as well as various MHC-II ligands. These new reagents will provide the basis for studying the interaction of LAG-3 with different MHC-II conformations and for evaluating the role of its interaction with FGL1.