Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate
Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate
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DOI：
10.1021/bi981616s
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发表时间：
1998-11-24
期刊：
影响因子：
2.9
通讯作者：
Remington, SJ
中科院分区：
文献类型：
--
作者：
Ormö, M;Bystrom, CE;Remington, SJ
大肠杆菌甘油激酶（GK）的三维结构与结合甘油在存在和不存在的变构调节剂的活性，果糖1，6-二磷酸（FBP），在3.2和3.0埃，提出。该分子属于P4（1）2（1）2空间群，结构用分子置换法解析。用良好的立体化学对模型进行了优化，最终R因子分别为21.1和21.9%。观察到单体的四聚体排列，其基本上与先前描述的建议的无活性四聚体II相同[Feese，M. D、Faber，H. R.，比斯特伦角E、Pettigrew，D. W.，和Remington，S. J.（1998）Structure（in press）]。然而，这种形式的晶体堆积是特别开放的，允许FBP结合位点被占据和识别。晶体学数据揭示了在两个甘氨酸-精氨酸环（残基234-236）之间形成的最不寻常类型的FBP结合位点，其中一半的结合位点由调节界面处的每个单体提供。FBP分子在非晶体学2倍轴上以两种相互排斥的模式结合，各占50%;因此，GK四聚体结合两个FBP分子。离子之间的相互作用的1-和6-磷酸的FBP和精氨酸236观察到除了氢键之间的相互作用的骨干酰胺的Gly 234和6-磷酸。未观察到蛋白质与呋喃糖环之间的接触。精氨酸236突变为丙氨酸，大大降低了由FBP抑制GK的程度，并降低了，但没有消除，FBP促进四聚体协会的能力。这些观察结果与先前表征的FBP抑制GK的机制一致，其中FBP既促进二聚体-四聚体组装又抑制四聚体。
The three-dimensional structures of Escherichia coli glycerol kinase (GK) with bound glycerol in the presence and absence of one of the allosteric regulators of its activity, fructose 1,6-bisphosphate (FBP), at 3.2 and 3.0 Angstrom, are presented. The molecule crystallized in space group P4(1)2(1)2, and the structure was solved by molecular replacement. The models were refined with good stereochemistry to final R-factors of 21.1 and 21.9%, respectively. A tetrameric arrangement of monomers was observed which was essentially identical to the proposed inactive tetramer II previously described [Feese, M. D., Faber, H. R., Bystrom, C. E., Pettigrew, D. W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal packing in this form was especially open, permitting the FBP binding site to be occupied and identified. The crystallographic data revealed a most unusual type of FBP binding site formed between two glycine-arginine loops (residues 234-236) where one-half of the binding site is donated by each monomer at the regulatory interface. The molecule of FBP binds in two mutually exclusive modes on a noncrystallographic 2-fold axis at 50% occupancy each; thus, a tetramer of GK binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of FBP and Arg 236 were observed in addition to hydrogen bonding interactions between the backbone amide of Gly 234 and the 6-phosphate. No contacts between the protein and the furanose ring were observed. Mutagenesis of Arg 236 to alanine drastically reduced the extent of inhibition of GK by FBP and lowered, but did not eliminate, the ability of FBP to promote tetramer association. These observations are consistent with the previously characterized mechanism of FBP inhibition of GK, in which FBP acts both to promote dimer-tetramer assembly and to inactivate the tetramers.