Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins
Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins
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DOI：
10.1101/785121
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发表时间：
2019-09
期刊：
影响因子：
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通讯作者：
Cecilia Piergentili;J. Ross;D. He;Kelly J. Gallagher;Will A. Stanley;Laurène Adam;C. Mackay;Kevin J. Waldron;David J. Clarke;J. Marles-Wright
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作者：
Cecilia Piergentili;J. Ross;D. He;Kelly J. Gallagher;Will A. Stanley;Laurène Adam;C. Mackay;Kevin J. Waldron;David J. Clarke;J. Marles-Wright
封装铁蛋白样蛋白属于普遍分布的铁蛋白超家族，其功能为铁解毒和储存系统。封装的铁蛋白具有独特的环形结构，并且必须与封装蛋白纳米笼结合以形成有效的铁储存，能够比经典铁蛋白容纳更多的铁。由于亚铁氧化酶催化位点和与其相邻的次级金属结合位点的组织差异，铁蛋白家族中铁氧化的催化机制仍然是一个悬而未决的问题。我们之前已经在红色红螺菌封装铁蛋白的内表面上确定了一个假定的金属结合位点，该位点位于双螺旋亚基之间的界面处且靠近亚铁氧化酶中心。在这里，我们提出了一项全面的结构和功能研究，通过酶测定、质谱和 X 射线晶体学来研究这个假定的铁进入位点的功能相关性。我们表明催化发生在亚铁氧化酶中心，并表明次级位点具有双重作用，即作为引导亚铁离子流向亚铁氧化酶中心的静电陷阱，同时充当保护亚铁氧化酶位点免受非同源抑制物质侵害的屏障。此外，将封装的铁蛋白限制在封装蛋白纳米笼内，在增强铁蛋白进行催化的能力的同时，不会影响第二位点的特定功能。
The Encapsulated ferritin-like proteins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. The encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question due to differences in organisation of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a putative metal binding site on the inner surface of Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase centre. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase centre and suggest a dual role for the secondary site as an electrostatic trap guiding ferrous ions toward the ferroxidase centre and, at the same time, acting as a barrier protecting the ferroxidase site against non-cognate inhibiting species. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ferritin ability to undergo catalysis, does not influence the specific function of the secondary site.