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Structural studies of the mechanisms for antigenic peptide binding of MHC class I molecule

MHC I 类分子抗原肽结合机制的结构研究

基本信息

批准号：
16590032
负责人：
KURIMOTO Eiji
金额：
$ 1.86万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590032/
关键词：
MHC NMR antigenic peptide empty refolding

项目摘要

1.Using an improved refolding method in the absence of an antigenic peptide, we have successfully prepared large quantities of empty MHC molecules.2. The ^1H^<-15>N HSQC spectrum of the empty MHC molecule whose heavy chain was labeled with ^<15>N exhibited peaks corresponding to the peptide-bound MHC molecule and those characteristic of random coiled states, suggesting coexistence of preserved and unfolded regions within the molecule. Using a selective labeling technique of distinct amino acid residues, it was revealed that α3 domain maintains a native tertiary structure whereas α 1 and α 2 domains, which form the antigen binding site, are partially unfolded in the absence of an antigenic peptide.3.We have assigned 87 % ^1H^<-15>N TROSY peaks originating from the backbone amide groups of β_2m by combined use of a deuterate labeling technique. Using the assigned NMR signals as spectroscopic probes, we evaluated the effects of dissociation of the antigenic peptide on the β_2m structure. Based on the NMR data, we clarified that only the hydrophobic cluster located just bellow the antigen binding pocket and its vicinity are perturbed upon dissociation of the antigenic peptide.4.We have identified the binding sites of LILRB1and LILRB2 on the β_2m subunit embedded in the MHC class I molecule using NMR chemical-shift-perturbation data.5.We analyzed the interaction between MHC class I molecules and molecular chaperones calnexin and calreticulin by means of surface plasmon resonance. It was revealed that calnexin and calreticulin bind to MHC molecules even without the modification with sugar chains and that the affinities of these chaperones for empty MHC molecules were slightly higher than those for peptide-bound MHC molecules, suggesting that they recognize denatured surface area of the empty MHC molecules.

1.在缺乏抗原肽的情况下，利用改进的重折叠方法，我们成功地制备了大量的空MHC分子.空<-15>MHC分子（其重链用^ N标记）的^1H ^ N HSQC光谱<15>显示出与肽结合的MHC分子相对应的峰，以及随机卷曲状态的特征峰，表明分子内存在保留区和未折叠区。采用不同氨基酸残基的选择性标记技术，发现α3结构域保持了天然的三级结构，而构成抗原结合位点的α 1和α 2结构域在缺乏抗原肽的情况下部分解折叠。3.结合氘标记技术，我们确定了87%的^1H^<-15>N TROSY峰来自β_2m的骨架酰胺基团。利用核磁共振信号作为光谱探针，研究了抗原肽解离对β_2m结构的影响。根据核磁共振数据，我们澄清了只有位于抗原结合口袋下方及其附近的疏水性簇在抗原肽解离时受到干扰。4.我们使用NMR化学位移鉴定了嵌入MHC I类分子中的β_2 m亚基上LILRB 1和LILRB 2的结合位点。5.利用表面等离子体共振技术分析了MHC I类分子与分子伴侣钙连接蛋白和钙网蛋白之间的相互作用。结果表明，即使没有糖链修饰，钙连接蛋白和钙网蛋白也与MHC分子结合，并且这些分子伴侣对空MHC分子的亲和力略高于肽结合的MHC分子，表明它们识别空MHC分子的变性表面区域。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d)

DOI：
10.1073/pnas.0605228103
发表时间：
2006-10-31
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Shiroishi, Mitsunori;Kuroki, Kimiko;Maenaka, Katsumi
通讯作者：
Maenaka, Katsumi

Structural basis for recognition of the non-classical MHC molecule HLA-G by the leukocyte Ig-like receptor B2(LILRBII/LIR2/ILT4/CD85d)

白细胞Ig样受体B2（LILRBII/LIR2/ILT4/CD85d）识别非经典MHC分子HLA-G的结构基础