Molecular Interactions Of Lymphoid Cell Receptors

淋巴细胞受体的分子相互作用

• 批准号: 10272044
• 负责人: David Margulies
• 金额: $ 75.6万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272044
• 关键词: Adaptive Immune System; Binding; Biological; C-terminal; CD8-Positive T-Lymphocytes; Cell Surface Receptors; Cells; Complex; Crystallization; Cysteine; Development; Diagnostic; Disulfides; Engineering; Event; Exhibits; Goals; HLA-B Antigens; Human; Immune; Immune system; Immunization; Immunologic Receptors; Innate Immune System; Laboratories; Ligands; Link; Lymphoid Cell; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Structure; Mus; Mutate; Mycobacterium tuberculosis; Peptide/MHC Complex; Peptides; Population; Positioning Attribute; Reagent; Receptor Cell; Roentgen Rays; Signal Transduction; Site; Stains; Structure; System; T cell response; T-Lymphocyte; TRAP Peptide; Therapeutic; Vaccines; Variant; Viral; base; cross reactivity; disulfide bond; insight; macromolecule; multiple myeloma M Protein; novel; novel vaccines; rBCG; receptor; tool

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.

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。此外，我们利用了几种策略来生成肽/MHC复合物，这些复合物利用二硫陷阱方法将肽变体与MHC分子共价连接。这些分子包括与各种截断肽连接的人HLA-B*44:05分子，以及人HLA-B*27:05和HLA-B*27:09分子。(2)与博士合作开展。Shihoko Komine-Aizawa和Mitsuo Honda研究了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MHC分子如H2-Kd结合。这些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细胞群的结构洞察。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。