Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: Evidence of conformational heterogeneity
Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: Evidence of conformational heterogeneity
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DOI：
10.1016/j.jmb.2003.10.022
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发表时间：
2003-12-12
影响因子：
5.6
通讯作者：
Polikarpov, I
中科院分区：
文献类型：
--
作者：
Aparicio, R;Ferreira, ST;Polikarpov, I
三磷酸酯异构酶（TIM）的活性位点环呈现出一种铰链盖运动，在两种明确的“开放”和“封闭”构象之间交替。到目前为止，封闭构象仅在具有底物类似物的蛋白质复合物中观察到。在这里，我们提出了第一个兔子肌肉载脂蛋白TIM结构，细化到1.5埃分辨率，其中活性位点环在酶的不同亚基中处于开放或封闭构象。在这里描述的封闭构象中，盖子环残基参与了稳定全息TIM结构特征的氢键，而在开环构象中观察到的化学相互作用与TIM的载子结构相似。在封闭构象中，在与活性位点残基形成氢键的投射配体原子位置上观察到许多水分子。结晶过程中使用的添加剂（DMSO和Tris分子和镁原子）在电子密度图中建模。然而，在活性位点和盖环附近没有观察到这些分子的特异性结合。为了进一步研究载脂蛋白酶的这种不寻常的封闭构象，确定了另外两个兔子肌肉的TIM结构，一个具有相同的晶体形式，另一个具有不同的晶体形式。这些结构只呈现开盖构象，表明闭合构象不能用晶体接触效应来解释。为了解释为什么在其中一个亚基中没有配体的情况下活性位点环是关闭的，在大量关于TIM酶动力学和反应机理的现有实验信息的支持下，与先前解决的TIM结构进行了广泛的比较。在TIM晶体中观察到的开盖和闭盖构象可能与该蛋白在溶液中的持续构象异质性有关。(C) 2003 Elsevier Ltd.版权所有。
The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5 Angstrom resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water, molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site-and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution. (C) 2003 Elsevier Ltd. All rights reserved.