Christodoulou Evangelos, Rypniewski Wojciech R, Vorgias Constantinos R E
National and Kapodistrian University of Athens, Faculty of Biology, Department of Biochemistry and Molecular Biology, Panepistimiopolis-Zographou, 157 84 Athens, Greece.
Extremophiles. 2003 Apr;7(2):111-22. doi: 10.1007/s00792-002-0302-7. Epub 2002 Dec 12.
The histone-like DNA-binding proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability. We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU protein from the hyper-thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C). The structural data presented clearly show that the HU Tmar has a fold similar to its thermophilic homologue HU from Bacillus stearothermophilus (HU Bst). Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar. Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme thermophilic protein HU Tmar to mesophilic, like HU Bsu. In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu. The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type. The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution. A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa. Our analysis points to this as the prime cause of the destabilization compared to the wild type. The three new structures were compared, together with the X-ray structure of a similar protein, HU Bst, with the aim of relating their structural properties and different thermal stability. The presented results show that the HU Tmar protein achieves its stability by employing a dual strategy. On the one hand, we observe local hydrophobic interactions, which stabilize the secondary structure elements, and on the other hand, electrostatic interactions between side chains.
类组蛋白DNA结合蛋白（HU）是研究影响热稳定性因素的一个便捷模型，因为它们具有相对简单、易于比较的结构，共同的功能，且存在于热稳定性差异很大的生物体中。我们报道了来自嗜热真细菌嗜热栖热菌（HU Tmar，Tm = 80.5℃）的HU蛋白在273K和100K时的高分辨率结构（1.53 Å）的测定结果。所呈现的结构数据清楚地表明，HU Tmar具有与其嗜热同源物来自嗜热脂肪芽孢杆菌的HU（HU Bst）相似的折叠结构。基于一级结构分析，以及对HU Bst（Tm = 61.6℃）和枯草芽孢杆菌（HU Bsu，Tm = 39.7℃）的突变分析结果，我们设计并产生了几个单突变和组合突变，以研究它们对重组HU Tmar热稳定性的影响。其中，三联体突变体HU Tmar - G15E/E34D/V42I（Tm = 35.9℃）已将极端嗜热蛋白HU Tmar转变为嗜温蛋白，类似于HU Bsu。为了分析HU Tmar的各种突变体，我们使点突变HU Tmar - E34D结晶，其中Glu34被Asp取代，类似于嗜温的HU Bsu。通过圆二色性测量，该突变体的Tm = 72.9℃，比野生型低7.6℃。HU Tmar - E34D的晶体结构在100K时测定，并在1.72 Å分辨率下进行精修。与野生型结构的比较清楚地表明，一个亚基的Glu34与另一个亚基的Thr13之间的两个氢键已被破坏，反之亦然。我们的分析指出，这是与野生型相比不稳定的主要原因。将这三个新结构与一种类似蛋白HU Bst的X射线结构进行了比较，目的是关联它们的结构特性和不同的热稳定性。所呈现的结果表明，HU Tmar蛋白通过采用双重策略实现其稳定性。一方面，我们观察到局部疏水相互作用，它稳定了二级结构元件，另一方面，是侧链之间的静电相互作用。
