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

1.利用一种改进的复性方法，在没有抗原肽的情况下，我们成功地制备了大量的空MHC分子。重链标记的空MHC分子的1H^-15&GT；N HSQC谱显示出与多肽结合的MHC分子相对应的峰，以及那些具有随机卷曲状态的峰，表明分子中存在保留区域和未折叠区域共存。利用不同氨基酸残基的选择性标记技术，发现α3结构域保持着天然的三级结构，而形成抗原结合部位的α1和α2结构域在缺乏抗原肽的情况下部分展开。3.我们结合氚标记技术对来自β_2m主链氨基的87%的峰进行了归属。用指定的核磁共振信号作为光谱探针，我们评估了抗原肽的解离对β_2m结构的影响。根据核磁共振数据，我们明确了只有位于抗原结合口袋及其附近的疏水簇会在抗原多肽解离时受到扰动。4.我们利用核磁共振化学位移微扰数据确定了LILRB1和LilrB2与嵌入在MHC I类分子中的β_2M亚基的结合位置。5.利用表面胞质共振技术分析了MHC I类分子与分子伴侣钙粘蛋白和钙网蛋白的相互作用。结果表明，即使在没有糖链修饰的情况下，钙粘蛋白和钙网蛋白也能与MHC分子结合，并且它们对空MHC分子的亲和力略高于与多肽结合的MHC分子，这表明它们识别空MHC分子的变性表面积。

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.