Wu Yan, Huo Chunheng, Ming Tinghong, Liu Yan, Su Chang, Qiu Xiaoting, Lu Chenyang, Zhou Jun, Li Ye, Zhang Zhen, Han Jiaojiao, Feng Ying, Su Xiurong
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo 315211, China.
College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315832, China.
Polymers (Basel). 2022 Dec 8;14(24):5378. doi: 10.3390/polym14245378.
Ferritin is widely acknowledged as a conservative iron storage protein found in almost all living kingdoms. (Selenka) is among the oldest echinoderm fauna and has unique regenerative potential, but the catalytic mechanism of iron oxidation in ferritin (AjFER) remains elusive. We previously identified several potential metal-binding sites at the ferroxidase center, the three- and four-fold channels in AjFER. Herein, we prepared AjFER, AjFER-E25A/E60A/E105A, AjFER-D129A/E132A, and AjFER-E168A mutants, investigated their structures, and functionally characterized these ferritins with respect to Fe uptake using X-ray techniques together with biochemical analytical methods. A crystallographic model of the AjFER-D129A/E132A mutant, which was solved to a resolution of 1.98 Å, suggested that the substitutions had a significant influence on the quaternary structure of the three-fold channel compared to that of AjFER. The structures of these ferritins in solution were determined based on the molecular envelopes of AjFER and its variants by small-angle X-ray scattering, and the structures were almost consistent with the characteristics of well-folded and globular-shaped proteins. Comparative biochemical analyses indicated that site-directed mutagenesis of metal-binding sites in AjFER presented relatively low rates of iron oxidation and thermostability, as well as weak iron-binding affinity, suggesting that these potential metal-binding sites play critical roles in the catalytic activity of ferritin. These findings provide profound insight into the structure-function relationships related to marine invertebrate ferritins.
铁蛋白被广泛认为是一种保守的铁储存蛋白，几乎存在于所有生物界。（海胆）是最古老的棘皮动物群之一，具有独特的再生潜力，但铁蛋白（AjFER）中铁氧化的催化机制仍然不清楚。我们之前在AjFER的铁氧化酶中心、三倍和四倍通道中确定了几个潜在的金属结合位点。在此，我们制备了AjFER、AjFER-E25A/E60A/E105A、AjFER-D129A/E132A和AjFER-E168A突变体，研究了它们的结构，并使用X射线技术和生化分析方法对这些铁蛋白的铁摄取功能进行了表征。AjFER-D129A/E132A突变体的晶体学模型分辨率为1.98 Å，表明与AjFER相比，这些取代对三倍通道的四级结构有显著影响。通过小角X射线散射，基于AjFER及其变体的分子包络确定了这些铁蛋白在溶液中的结构，这些结构几乎与折叠良好的球状蛋白的特征一致。比较生化分析表明，AjFER中金属结合位点的定点诱变呈现出相对较低的铁氧化速率和热稳定性，以及较弱的铁结合亲和力，这表明这些潜在的金属结合位点在铁蛋白的催化活性中起关键作用。这些发现为深入了解与海洋无脊椎动物铁蛋白相关的结构-功能关系提供了深刻见解。
