Vyas Nand K, Vyas Meenakshi N, Chervenak Mary C, Johnson Margaret A, Pinto B Mario, Bundle David R, Quiocho Florante A
Verna and Marrs Mclean Department of Biochemistry and Molecular Biology and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA.
Biochemistry. 2002 Nov 19;41(46):13575-86. doi: 10.1021/bi0261387.
The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.
通过对Fab结构域及其与两个抗原片段（一个五糖Rha A-Rha B-Rha C-GlcNAc D-Rha A'和一个修饰的三糖Rha B-Rha C-GlcNAc D，其中Rha C*缺少C2-OH基团）的两种复合物进行晶体学分析，研究了福氏志贺菌O-多糖（由重复单元ABCD [-->2)-α-L-Rhap-(1-->2)-α-L-Rhap-(1-->3)-α-L-Rhap-(1-->3)-β-D-GlcpNAc-(1-->]组成）被单克隆抗体SYA/J6（IgG3，κ）的抗原识别情况。这些复合物结构是针对周期性线性杂多糖的Fab的首个此类结构，揭示了一个结合位点凹槽（在V(H)和V(L)结构域之间），该凹槽与五糖的所有糖残基进行极性和非极性接触。Fab的主链和侧链原子均用于配体结合。CDR H2的Glu H50的带电侧链与寡糖的GlcNAc形成关键氢键。修饰的三糖比五糖埋藏更深且契合更紧密。它与Fab形成的接触（约75个）与五糖一样多，包括相同数量的氢键（八个，其中四个相同）。它还比五糖参与更多的疏水相互作用。这三个有利于三糖结合的特征与观察到的三糖复合物比五糖复合物更紧密一致。热力学数据表明，天然的三糖至五糖的结合自由能在6.8 - 7.4 kcal mol(-1)范围内，并且与单个羟基形成的氢键中，除一个外，其余提供的能量不超过约0.7 kcal mol(-1)。这些数据还表明，疏水相互作用对结合有显著贡献，并且随着天然表位在三糖、四糖、五糖系列中变大，熵对自由能的贡献变得占主导地位。
