Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR
Picometer Resolution Structure of the Coordination Sphere in the Metal-Binding Site in a Metalloprotein by NMR
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DOI：
10.1021/jacs.0c07339
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发表时间：
2020-09-30
影响因子：
15
通讯作者：
Pintacuda, Guido
中科院分区：
文献类型：
--
作者：
Bertarello, Andrea;Benda, Ladislav;Pintacuda, Guido
我们对化学的大部分理解来自于用单晶x射线衍射获得的原子级结构。小型有机金属或配位配合物的x射线结构中的金属中心通常非常明确，其位置误差在10(-4)-10(-5)a的数量级上。高精度地确定金属配位几何对于理解金属中心反应性至关重要，因为即使很小的结构变化也会极大地改变金属活性。相比之下，蛋白质中x射线结构的分辨率通常限制在10（-1）埃的数量级。这种分辨率通常不足以为蛋白质中的金属位点建立精确的结构-活性关系，因为位置的不确定性可以覆盖给定类型的金属配合物的键长和键角的所有已知范围。在这里，我们介绍了一种新的方法，可以通过结合魔角旋转（MAS）核磁共振（NMR）光谱，定制射频（RF）照射方案和计算方法，从粉末样品中确定金属蛋白活性位点的高清结构。这使我们能够克服顺磁性蛋白质中的“盲球”，并观察和分配直接配位金属中心的配体的H-1， C-13和N-15共振。我们通过以0.7 pm的精度测定人类超氧化物歧化酶1 （SOD）核心Co-II配位球结构中的键长来说明该方法。所得到的配位几何结构解释了这些蛋白质中Co-II/Zn-II中心的非反应性，这使得它们能够发挥纯粹的结构作用。
Most of our understanding of chemistry derives from atomic-level structures obtained with single-crystal X-ray diffraction. Metal centers in X-ray structures of small organometallic or coordination complexes are often extremely well-defined, with errors in the positions on the order of 10(-4)-10(-5) A. Determining the metal coordination geometry to high accuracy is essential for understanding metal center reactivity, as even small structural changes can dramatically alter the metal activity. In contrast, the resolution of X-ray structures in proteins is limited typically to the order of 10(-1) angstrom. This resolution is often not sufficient to develop precise structure-activity relations for the metal sites in proteins, because the uncertainty in positions can cover all of the known ranges of bond lengths and bond angles for a given type of metal complex. Here we introduce a new approach that enables the determination of a high-definition structure of the active site of a metalloprotein from a powder sample, by combining magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, tailored radio frequency (RF) irradiation schemes, and computational approaches. This allows us to overcome the "blind sphere" in paramagnetic proteins, and to observe and assign H-1, C-13, and N-15 resonances for the ligands directly coordinating the metal center. We illustrate the method by determining the bond lengths in the structure of the Co-II coordination sphere at the core of human superoxide dismutase 1 (SOD) with 0.7 pm precision. The coordination geometry of the resulting structure explains the nonreactive nature of the Co-II/Zn-II centers in these proteins, which allows them to play a purely structural role.