Geremia Silvano, Di Costanzo Luigi, Randaccio Lucio, Engel Donald E, Lombardi Angela, Nastri Flavia, DeGrado William F
Centre of Excellence in Biocrystallography, Department of Chemical Science, University of Trieste, Via L. Giorgieri 1, I-34127 Trieste, Italy.
J Am Chem Soc. 2005 Dec 14;127(49):17266-76. doi: 10.1021/ja054199x.
The de novo protein DF1 is a minimal model for diiron and dimanganese metalloproteins, such as soluble methane monooxygenase. DF1 is a homodimeric four-helix bundle whose dinuclear center is formed by two bridging Glu side chains, two chelating Glu side chains, and two monodentate His ligands. Here, we report the di-Mn(II) and di-Co(II) derivatives of variants of this protein. Together with previously solved structures, 23 crystallographically independent four-helix bundle structures of DF1 variants have been determined, which differ in the bound metal ions and size of the active site cavity. For the di-Mn(II) derivatives, as the size of the cavity increases, the number and polarity of exogenous ligands increases. This collection of structures was analyzed to determine the relationship between protein conformation and the geometry of the active site. The primary mode of backbone movement involves a coordinated tilting and sliding of the first helix in the helix-loop-helix motif. Sliding depends on crystal-packing forces, the steric bulk of a critical residue that determines the dimensions of the active site access cavity, and the intermetal distance. Additionally, a torsional motion of the bridging carboxylates modulates the intermetal distance. This analysis provides a critical evaluation of how conformation, flexibility, and active site accessibility affect the geometry and ligand-binding properties of a metal center. The geometric parameters defining the DF structures were compared to natural diiron proteins; DF proteins have a restricted active site cavity, which may have implications for substrate recognition and chemical stability.
从头合成蛋白DF1是二铁和二锰金属蛋白（如可溶性甲烷单加氧酶）的一个最小模型。DF1是一个同二聚体四螺旋束，其二核中心由两条桥连的谷氨酸侧链、两条螯合的谷氨酸侧链和两条单齿组氨酸配体形成。在此，我们报道了该蛋白变体的二价锰（II）和二价钴（II）衍生物。连同之前解析的结构，已确定了DF1变体的23个晶体学独立的四螺旋束结构，这些结构在结合的金属离子和活性位点腔的大小方面有所不同。对于二价锰（II）衍生物，随着腔的大小增加，外源配体的数量和极性增加。对这一系列结构进行了分析，以确定蛋白质构象与活性位点几何形状之间的关系。主链运动的主要模式涉及螺旋-环-螺旋基序中第一个螺旋的协同倾斜和滑动。滑动取决于晶体堆积力、决定活性位点进入腔尺寸的关键残基的空间体积以及金属间距离。此外，桥连羧酸盐的扭转运动调节金属间距离。该分析对构象、灵活性和活性位点可及性如何影响金属中心的几何形状和配体结合特性进行了关键评估。将定义DF结构的几何参数与天然二铁蛋白进行了比较；DF蛋白具有受限的活性位点腔，这可能对底物识别和化学稳定性有影响。
