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。对于上面提到的项目(1)，我们已经开发了一种产生二硫键捕获的多肽/MHC-I分子的策略，并已经表达、复性、纯化和结晶了其中的几个。特别是，我们研究了H2-DD分子，其中结合裂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多肽重新折叠这些分子。这些化合物的X-射线晶体结构证实，二硫键捕获的多肽稳定地固定在多肽结合槽中。用截短肽产生的这些分子的版本表明，这些分子在部分肽结合槽中是肽稳定的，但在截断位置处是松弛的。结合研究表明，这些分子应该可以绘制与各种细胞和病毒伴侣相互作用的位置图。使用一种略有不同的方法，我们已经产生了几种具有新的链内二硫键的MHC-I分子，特别是H2-DD和H2-LD。虽然我们最初预计这些分子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分子结合的特定多肽有关。进一步的研究不仅包括稳定性，还包括生物活性和作为四聚体染色试剂的用途。此外，我们还利用了几种策略来产生多肽/MHC复合体，利用二硫键陷阱方法将多肽变体共价连接到MHC分子。这些分子包括与各种截短肽相连的人类人类白细胞抗原-B*44：05分子，以及人类人类白细胞抗原-B*27：05和人类白细胞抗原B*27：09分子。For(2)与Shihoko Komine-Aizawa博士和Mitsuo Honda博士合作，探索了CD8 T细胞对编码结核分枝杆菌Ag85B蛋白的重组卡介苗免疫小鼠的反应。我们鉴定了几个H2-KD限制性的Ag85B多肽(YYQSGLSIV和YQSGLSIVM)，并对它们的X射线结构进行了结晶和测定。这些结构表明，变异肽可以以新的构象与像H2-KD这样的MHC分子结合。对这些的分析提供了对由新疫苗启动的交叉反应和非交叉反应T细胞群体的结构性洞察。肽/H2-KD四聚体识别由Ag85B疫苗诱导的两个不同的T细胞群。特别是，YQSGLSIVM肽以非正则取向结合，其C末端残基延伸到肽结合槽之外。

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.