Xu G Y, Ong E, Gilkes N R, Kilburn D G, Muhandiram D R, Harris-Brandts M, Carver J P, Kay L E, Harvey T S
Department of Medical Genetics, University of Toronto, Ontario, Canada.
Biochemistry. 1995 May 30;34(21):6993-7009.
Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures. The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements. Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution. While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set. The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation. CBDcex forms an extensive beta-sheet structure with a beta-barrel fold. Titration with cellohexaose, [beta-D-glucopyranosyl-(1,4)]5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding. Analysis of the structure shows that these residues are adjacent in space and exposed to solvent. Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family.
多维、多核核磁共振光谱结合动态模拟退火已被用于确定来自纤维单胞菌（Cellulomonas fimi）的β-1,4-聚糖酶Cex的110个氨基酸的纤维素结合结构域（CBDcex）的结构。一个包含1795个质子间NOE衍生约束、50个φ角、34个χ1角和106个氢键约束的实验数据集被用于计算20个最终结构。当拟合到二级结构元件上时，计算得到的结构对于主链原子而言，相对于平均结构的平均均方根（rms）偏差为0.41 Å，对于所有重原子而言为0.67 Å。色谱法、超速离心和15N NMR弛豫实验表明，CBDcex在溶液中是二聚体。虽然跨二聚体界面测量NOE的尝试未成功，但采用了一种计算策略来生成与推导数据集一致的二聚体结构。二聚体计算结果表明，虽然在二聚体环境中产生的单体拓扑结构可能是可变的，但单体中存在的二级结构元件和侧链的相对位置在二聚体形成时得以恢复。CBDcex形成具有β-桶状折叠的广泛β-片层结构。用纤维六糖，即[β-D-吡喃葡萄糖基-(1,4)]5-D-葡萄糖进行滴定，确定Trp 54和72参与纤维素结合。结构分析表明，这些残基在空间上相邻且暴露于溶剂中。与其他相邻的亲水性残基一起，这些残基形成一个碳水化合物结合裂隙，这似乎是同一家族所有CBD的共同特征。
