Department of Chemistry, Macalester College, 1600 Grand Avenue, Saint Paul, MN, 55105, USA.
J Biol Inorg Chem. 2023 Aug;28(5):485-494. doi: 10.1007/s00775-023-02002-4. Epub 2023 Jun 2.
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 Zn-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.
通过金属离子取代来调节金属蛋白酶的结构和功能，可能构成了金属离子毒性和/或金属介导的功能调控的分子基础。X 连锁凋亡抑制蛋白（XIAP）是一种需要锌才能保持正确结构和功能的金属蛋白酶。除了作为凋亡调节剂的作用外，XIAP 还与铜稳态有关。鉴于铜和锌的配位偏好相似，研究 XIAP 与铜相互作用时的结构和功能具有重要意义。XIAP 的 Really Interesting New Gene（RING）结构域代表了一类锌指蛋白，它们利用双核锌结合基序来维持正确的结构和泛素连接酶功能。在此，我们报告了铜（I）与 XIAP 的 Zn-RING 结构域结合的特征。监测铜-硫醇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，并且铜在热力学上优先于锌。在存在 Zn（II）特异性染料 Mag-Fura2 的情况下重复实验表明，即使存在谷胱甘肽，Cu（I）的加入也会导致蛋白质中 Zn（II）的排出。铜取代锌结合位点后，RING 结构域的二聚体结构丢失，这是其泛素连接酶活性的要求，这通过尺寸排阻色谱很容易观察到。这些结果为铜对 RING 功能的调节提供了分子基础，并为越来越多的描述 Cu（I）对锌金属蛋白酶结构和功能影响的文献增添了内容。
