Hart P J, Nersissian A M, Herrmann R G, Nalbandyan R M, Valentine J S, Eisenberg D
UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, University of California 90095, USA.
Protein Sci. 1996 Nov;5(11):2175-83. doi: 10.1002/pro.5560051104.
Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.
星蓝蛋白是蓝色（I型）铜糖蛋白，在光谱和电子转移特性方面与铜氧化还原蛋白家族的其他成员不同。到目前为止，星蓝蛋白的结构测定一直未能实现。在此，我们报告了重组黄瓜星蓝蛋白109个氨基酸的非糖基化铜结合结构域的三维晶体结构，其精修分辨率达到1.6 Å。50 - 1.6 Å之间所有18488个反射（σ > 0）的晶体学R值为0.195。整体折叠结构由两个β折叠片组成，每个β折叠片都有四条β链。在β链之间的环区域发现了两个α螺旋。β折叠片形成了一个β三明治结构，类似于其他铜氧化还原蛋白中的结构，但某些特征与质体蓝素和天青蛋白等蛋白质不同，即β桶更扁平，有一个额外的N端α螺旋，并且铜结合位点更容易被溶剂接触。蛋白质铜结合端存在二硫键，这证实黄瓜星蓝蛋白具有植物蓝蛋白样的折叠结构。与铜配位的配体是两个组氨酸、一个半胱氨酸和一个谷氨酰胺，后者取代了单核蓝色铜蛋白中常见的甲硫氨酸。Cu - Gln键是迄今为止在结构表征的I型铜蛋白中观察到的最短的轴向配体键距离之一。星蓝蛋白独特的光谱特性和电子转移反应性与其他特征明确的铜氧化还原蛋白有显著差异，这可以通过其更暴露的铜位点、独特的氨基酸配体组成以及近乎四面体的配体几何结构来解释。文中还讨论了黄瓜星蓝蛋白分子上可能参与与假定氧化还原伙伴相互作用的表面特征。
