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），我们已经开发了一种用于产生二硫键捕获的肽/MHC-I分子的策略，并且已经表达、重折叠、纯化和结晶了其中的几种。特别是，我们已经研究了H2-Dd分子，其中结合裂缝中的残基已突变为半胱氨酸，并在适当的位置用含有半胱氨酸的肽重新折叠这些分子。这些的X射线晶体结构证实，二硫键捕获的肽稳定地固定在肽结合沟中。用截短的肽产生的这些版本表明，这些分子在肽结合沟的一部分中是肽稳定的，但在截短的位置中是松弛的。结合研究表明，这些分子应该允许映射与各种细胞和病毒伴侣的相互作用位点。使用稍微不同的方法，我们已经产生了具有新的链内二硫键的MHC-I分子的几种变体，特别是H2-Dd和H2-Ld。虽然我们最初期望这些具有更高的热稳定性，但正在进行的研究表明，稳定性也与这些MHC分子结合的特定肽有关。进一步的研究，不仅调查的稳定性，但作为四聚体染色试剂的生物活性和实用性正在进行中。此外，我们已经利用了几种策略，用于产生肽/MHC复合物，共价连接肽变体的MHC分子，利用二硫化物陷阱的方法。这些分子包括与各种截短肽连接的人HLA-B*44：05分子，以及人HLA-B*27：05和HLA-B*27：09分子。对于（2），与Shihoko Komine-Aizawa博士和Mitsuo本田博士合作进行，探索了用编码M的Ag 85 B蛋白的重组BCG疫苗免疫小鼠的CD 8 T细胞应答。结核几个H2-Kd-限制性的Ag 85 B肽被确定（YYQSGLSIV和YQSGLSIVM），我们已经结晶和确定这些的X-射线结构。这些结构表明，变体肽可以以新的构象与MHC分子如H2-Kd结合。对这些的分析提供了对新疫苗引发的交叉反应性和非交叉反应性T细胞群体的结构洞察。肽/H2-Kd四聚体鉴定由Ag 85 B疫苗引发的两种不同的T细胞群。特别地，YQSGLSIVM肽以非典型取向结合，其C-末端残基延伸超过肽结合沟。

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 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. In addition, we have taken advantage of several strategies for generating peptide/MHC complexes that covalently link peptide variants to MHC molecules, exploiting a disulfide trap approach. These molecules include the human HLA-B*44:05 molecule linked to various truncated peptides, as well as the human HLA-B*27:05 and HLA-B*27:09 molecules. For (2) carried out collaboratively with Drs. 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. Analysis of these offers 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.