The Effect of Asp-His-Ser/Thr-Trp Tetrad on the Thermostability of WD40-Repeat Proteins
The Effect of Asp-His-Ser/Thr-Trp Tetrad on the Thermostability of WD40-Repeat Proteins
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DOI：
10.1021/bi101321y
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发表时间：
2010-11-30
期刊：
影响因子：
2.9
通讯作者：
Wu, Yun-Dong
中科院分区：
文献类型：
--
作者：
Wu, Xian-Hui;Chen, Rong-Chang;Wu, Yun-Dong
我们最近发现，Asp-His-Ser/Thr-Trp氢键四联体广泛而独特地存在于WD40-Repeat蛋白中。WDR5蛋白是一个7个WD40重复的螺旋桨，有5个这样的四分体。为了探讨四分体对WD40-Repeat蛋白结构和稳定性的影响，我们分离了野生型WDR5及其七个涉及四分体残基替换的突变体。用X射线衍射法测定了野生型WDR5及其三个突变体的晶体结构。发现四分体残基的突变不会改变基本的结构特征。用圆二色谱研究了野生型和7个突变型蛋白在盐酸胍变性剂作用下的变性特征，以确定这些蛋白的折叠自由能。野生型和S62A、S146A、S188A、D192E、W330F、W330Y和D324E突变体的折叠自由能分别为-11.6、-2.7、-3.1、-2.9、-3.6、-7.1、-7.0和-7.5千卡/摩尔。这表明：(1)这些氢键网络中的氢键非常强；(2)每个氢键四分体为蛋白质提供了超过12千卡/摩尔的稳定性；因此，任何一个四分体的去除都会导致蛋白质的去折叠；(3)由于有五个四分体，如果没有四分体，蛋白质肯定处于高度不稳定的状态，这可能与其生物学功能有关。
We recently found that Asp-His-Ser/Thr-Trp hydrogen-bonded tetrads are widely and uniquely present in the WD40-repeat proteins. WDR5 protein is a seven WD40-repeat propeller with five such tetrads. To explore the effect of the tetrad on the structure and stability of WD40-repeat proteins, the wild-type WDR5 and its seven mutants involving the substitutions of tetrad residues have been isolated. The crystal structures of the wild-type WDR5 and its three WDR5 mutants have been determined by X-ray diffraction method. The mutations of the tetrad residues are found not to change the basic structural features. The denaturing profiles of the wild type and the seven mutants with the use of denaturant guanidine hydrochloride have been studied by circular dichroism spectroscopy to determine the folding free energies of these proteins. The folding free energies of the wild type and the S62A, S146A, S188A, D192E, W330F, W330Y, and D324E mutants are measured to be about -11.6, -2.7, -3.1, -2.9, -3.6, -7.1, -7.0, and -7.5 kcal/mol, respectively. These suggest that (1) the hydrogen bonds in these hydrogen bond networks are unusually strong; (2) each hydrogen-bonded tetrad provides over 12 kcal/mol stability to the protein; thus, the removal of any single tetrad would cause unfolding of the protein; (3) since there are five tetrads, the protein must be in a highly unstable state without the tetrads, which might be related to its biological functions.