Response of a designed metalloprotein to changes in metal ion coordination, exogenous ligands, and active site volume determined by X-ray crystallography
Response of a designed metalloprotein to changes in metal ion coordination, exogenous ligands, and active site volume determined by X-ray crystallography
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DOI：
10.1021/ja054199x
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发表时间：
2005-12-14
影响因子：
15
通讯作者：
DeGrado, WF
中科院分区：
文献类型：
--
作者：
Geremia, S;Di Costanzo, L;DeGrado, WF
从头蛋白DF1是二铁和二锰金属蛋白的最小模型，如可溶性甲烷单加氧酶。DF1是一个同源二聚体四螺旋束，其双核中心由两个桥联的Glu侧链、两个螯合的Glu侧链和两个单齿的His配体组成。在这里，我们报告了该蛋白质变体的di-Mn(II)和di-Co(II)衍生物。结合已有的结构，确定了23个DF1异构体的结晶学上独立的四螺旋束状结构，它们的结合金属离子和活性中心空穴的大小不同。对于di-Mn(II)衍生物，随着空腔尺寸的增大，外源配体的数量和极性增加。对这组结构进行分析，以确定蛋白质构象与活性部位几何形状之间的关系。脊椎运动的主要方式包括螺旋-环-螺旋模体中第一个螺旋的协调倾斜和滑动。滑动取决于晶体堆积力、决定活性中心访问腔尺寸的关键残基的空间体积以及金属间距离。此外，桥联羧酸盐的扭转运动调节了金属间的距离。这一分析提供了对构象、灵活性和活性中心可访问性如何影响金属中心的几何结构和配体结合性质的关键评估。将定义DF结构的几何参数与天然双铁蛋白进行了比较；DF蛋白有一个受限的活性部位空腔，这可能对底物识别和化学稳定性有影响。
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.