Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions
Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions
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DOI：
10.1007/s00775-023-02002-4
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发表时间：
2023-06
期刊：
影响因子：
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通讯作者：
Kathryn E. Splan;Sylvia R Choi;Ruth E Claycomb;Isaiah K. Eckart-Frank;Shreya Nagdev;Madeline E Rodemeier
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文献类型：
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作者：
Kathryn E. Splan;Sylvia R Choi;Ruth E Claycomb;Isaiah K. Eckart-Frank;Shreya Nagdev;Madeline E Rodemeier
通过金属离子取代调节金属蛋白的结构和功能可能构成金属离子毒性和/或金属介导的功能控制的分子基础。X-连锁凋亡抑制蛋白（XIAP）是一种金属蛋白，需要锌的适当结构和功能。除了其作为细胞凋亡调节剂的作用之外，XIAP还涉及铜稳态。考虑到铜和锌的类似配位偏好，研究XIAP结构和与铜相互作用后的功能是相关的。XIAP的真正有趣的新基因（RING）结构域是一类锌指蛋白的代表，其利用双核锌结合基序来维持适当的结构和泛素连接酶功能。在此，我们报告了铜（I）结合到XIAP的Zn 2-RING结构域的表征。监测铜-硫醇盐相互作用的电子吸收研究表明，XIAP的RING结构域结合5-6个Cu（I）离子，并且铜相对于锌在化学上是优选的。在Zn（II）特异性染料Mag-Fura 2的存在下重复实验表明，Cu（I）的加入导致Zn（II）从蛋白质中排出，即使在谷胱甘肽的存在下也是如此。通过尺寸排阻色谱法容易观察到RING结构域的二聚体结构的损失，这是其泛素连接酶活性的要求，在锌结合位点处的铜取代后。这些结果为铜调节RING功能提供了分子基础，并为描述Cu（I）对锌金属蛋白结构和功能的影响的越来越多的文献提供了补充。
Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper–thiolate interactions demonstrate that the RING domain of XIAP binds 5–6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.Graphical abstract