Eklund H, Ingelman M, Söderberg B O, Uhlin T, Nordlund P, Nikkola M, Sonnerstam U, Joelson T, Petratos K
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala.
J Mol Biol. 1992 Nov 20;228(2):596-618. doi: 10.1016/0022-2836(92)90844-a.
The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.
野生型噬菌体T4谷氧还蛋白（早期称为硫氧还蛋白）氧化形式的结构已在单斜晶系晶体中以2.0 Å分辨率进行了精修，晶体学R因子为0.209。一种突变型T4谷氧还蛋白能形成质量更好的正交晶体。该突变体的结构已通过分子置换法解析，并在1.45 Å分辨率下精修至R值为0.175。在这种突变型谷氧还蛋白中，活性位点残基Val15和Tyr16分别被Gly和Pro取代，以模拟大肠杆菌硫氧还蛋白。野生型蛋白的主链构象在单斜晶体不对称单元中的两个独立测定分子中相似。另一方面，由于晶体中的异源堆积相互作用，两个分子的侧链构象差异很大。突变型蛋白的结构与野生型蛋白非常相似，除了突变位置和参与晶体接触的部分。Cys14和Cys17之间的活性位点二硫键位于α1螺旋的第一个转角处。这些残基的扭转角与大肠杆菌硫氧还蛋白的相似。二硫键周围的扭转角小于二硫键通常观察到的角度：野生型谷氧还蛋白分子A和B以及突变型谷氧还蛋白分别为58度、67度和67度。T4谷氧还蛋白中二硫半胱氨酸的每个硫原子与一个主链氮原子形成氢键。活性位点一侧被半胱氨酸残基的β碳原子以及残基7、9、21和33的侧链屏蔽，免受溶剂影响。从相反一侧看，有一个裂缝，Cys14的硫原子可进入，在还原反应的初始步骤中可被亲核硫醇盐离子攻击。
