Casset F, Peters T, Etzler M, Korchagina E, Nifant'ev N, Pérez S, Imberty A
Institut National de la Recherche Agronomique, Nantes, France.
Eur J Biochem. 1996 Aug 1;239(3):710-9. doi: 10.1111/j.1432-1033.1996.0710u.x.
The present study is concerned with the elucidation of the conformation of the blood group A trisaccharide (alpha-D-GalNAc(1-3)[alpha-L-Fuc(1-->2)] beta-D-Gal-O-R) in the combining site of Dolichos biflorus seed lectin by use of 400-MHz and 600-MHz NMR spectroscopy. D. biflorus lectin displays a unique specificity for GalNAc residues. It occurs in solution as a tetrameric assembly having a molecular mass of 110 kDa, with two carbohydrate-binding sites per molecule. First, NOE build-up curves were obtained for the free blood group A trisaccharide from one-dimensional transient NOE experiments. Simulated NOE build-up curves were constructed from an ensemble of low-energy conformers derived from previous investigations. The comparison of theoretical and experimental data indicates that an equilibrium between two families of low-energy conformers most likely reflects the solution behavior of the trisaccharide in solution. Two-dimensional transferred NOE and rotating-frame enhancements (ROE) were subsequently measured for the trisaccharide complexed with the D. biflorus seed lectin. In addition to the NOEs observed for the free trisaccharide, the transferred NOESY spectrum showed several new NOEs that were identified as spin diffusion using a rotating-frame NOESY (ROESY) experiment. Experimental interglycosidic transferred nuclear Overhauser effect (TRNOE) build-up curves were compared to theoretical curves calculated for both low-energy conformers located in the D. biflorus lectin-binding site. Calculations of theoretical TRNOE were performed using a combination of the full relaxation matrix and the protein-ligand exchange matrix. Comparison between experimental and simulated TRNOE volumes leads to the conclusion that one conformation of blood group A trisaccharide is selected upon binding by D. biflorus lectin.
本研究旨在通过使用400兆赫和600兆赫核磁共振光谱来阐明血型A三糖（α-D-氨基半乳糖（1→3）[α-L-岩藻糖（1→2）]β-D-半乳糖-O-R）在双花扁豆种子凝集素结合位点的构象。双花扁豆凝集素对氨基半乳糖残基表现出独特的特异性。它在溶液中以分子量为110 kDa的四聚体形式存在，每个分子有两个碳水化合物结合位点。首先，通过一维瞬态核Overhauser效应实验获得游离血型A三糖的核Overhauser效应积累曲线。模拟的核Overhauser效应积累曲线是根据先前研究得出的低能构象体集合构建的。理论数据与实验数据的比较表明，两个低能构象体家族之间的平衡最有可能反映三糖在溶液中的溶液行为。随后对与双花扁豆种子凝集素复合的三糖进行二维转移核Overhauser效应和旋转框架增强（ROE）测量。除了游离三糖观察到的核Overhauser效应外，转移核Overhauser效应光谱还显示了几个新的核Overhauser效应，这些效应通过旋转框架核Overhauser效应光谱（ROESY）实验被鉴定为自旋扩散。将实验性糖苷间转移核Overhauser效应（TRNOE）积累曲线与为位于双花扁豆凝集素结合位点的两种低能构象体计算的理论曲线进行比较。使用全弛豫矩阵和蛋白质-配体交换矩阵的组合进行理论TRNOE的计算。实验和模拟的TRNOE体积之间的比较得出结论，血型A三糖的一种构象在与双花扁豆凝集素结合时被选择。
