Noble M E, Zeelen J P, Wierenga R K
European Molecular Biology Laboratory, Heidelberg, Germany.
Proteins. 1993 Aug;16(4):311-26. doi: 10.1002/prot.340160402.
The structure of trypanosomal triosephosphate isomerase (TIM) has been solved at a resolution of 2.1A in a new crystal form grown at pH 8.8 from PEG6000. In this new crystal form (space group C2, cell dimensions 94.8 A, 48.3 A, 131.0 A, 90.0 degrees, 100.3 degrees, 90.0 degrees), TIM is present in a ligand-free state. The asymmetric unit consists of two TIM subunits. Each of these subunits is part of a dimer which is sitting on a crystallographic twofold axis, such that the crystal packing is formed from two TIM dimers in two distinct environments. The two constituent monomers of a given dimer are, therefore, crystallographically equivalent. In the ligand-free state of TIM in this crystal form, the two types of dimer are very similar in structure, with the flexible loops in the "open" conformation. For one dimer (termed molecule-1), the flexible loop (loop-6) is involved in crystal contacts. Crystals of this type have been used in soaking experiments with 0.4 M ammonium sulphate (studied at 2.4 A resolution), and with 40 microM phosphoglycolohydroxamate (studied at 2.5 A resolution). It is found that transfer to 0.4 M ammonium sulphate (equal to 80 times the Ki of sulphate for TIM), gives rise to significant sulphate binding at the active site of one dimer (termed molecule-2), and less significant binding at the active site of the other. In neither dimer does sulphate induce a "closed" conformation. In a mother liquor containing 40 microM phosphoglycolohydroxamate (equal to 10 times the Ki of phosphoglycolohydroxamate for TIM), an inhibitor molecule binds at the active site of only that dimer of which the flexible loop is free from crystal contacts (molecule-2). In this dimer, it induces a closed conformation. These three structures are compared and discussed with respect to the mode of binding of ligand in the active site as well as with respect to the conformational changes resulting from ligand binding.
在pH 8.8条件下由聚乙二醇6000培养出的一种新晶型中，锥虫磷酸丙糖异构酶（TIM）的结构已通过分辨率为2.1埃的X射线晶体学方法解析出来。在这种新晶型（空间群C2，晶胞参数为94.8埃、48.3埃、131.0埃，α = 90.0°，β = 100.3°，γ = 90.0°）中，TIM以无配体状态存在。不对称单元由两个TIM亚基组成。每个亚基都是位于晶体学二重轴上的二聚体的一部分，这样晶体堆积是由处于两种不同环境中的两个TIM二聚体形成的。因此，给定二聚体的两个组成单体在晶体学上是等价的。在这种晶型的TIM无配体状态下，两种类型的二聚体在结构上非常相似，其柔性环处于“开放”构象。对于一个二聚体（称为分子1），柔性环（环6）参与晶体接触。这种类型的晶体已用于0.4 M硫酸铵的浸泡实验（在2.4埃分辨率下研究）以及40 μM磷酸甘油羟肟酸的浸泡实验（在2.5埃分辨率下研究）。研究发现，转移到0.4 M硫酸铵（相当于硫酸对TIM的解离常数Ki的80倍）后，一个二聚体（称为分子2）的活性位点会发生显著的硫酸根结合，而另一个二聚体的活性位点结合则不太明显。在这两个二聚体中，硫酸根都不会诱导出“封闭”构象。在含有40 μM磷酸甘油羟肟酸（相当于磷酸甘油羟肟酸对TIM的解离常数Ki的10倍） 的母液中，抑制剂分子仅结合在柔性环不参与晶体接触的那个二聚体（分子2）的活性位点上。在这个二聚体中，它会诱导出封闭构象。本文比较并讨论了这三种结构在活性位点配体结合模式以及配体结合导致的构象变化方面的情况。
