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Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions

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Abstract

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.

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Acknowledgements

This work was supported by the National Science Foundation (#CHE-1807773 to K.E.S.) and Macalester College.

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  1. Department of Chemistry, Macalester College, 1600 Grand Avenue, Saint Paul, MN, 55105, USA

    Kathryn E. Splan, Sylvia R. Choi, Ruth E. Claycomb, Isaiah K. Eckart-Frank, Shreya Nagdev & Madeline E. Rodemeier

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  1. Kathryn E. Splan
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  2. Sylvia R. Choi
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Correspondence to Kathryn E. Splan.

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775_2023_2002_MOESM1_ESM.pdf

Supplementary file1 (PDF 681 KB) Supplementary Material: Full sequence of XIAP-RING-W, mass spectral data, uncorrected absorption spectra, spectral response of Magfura-2 to Cu(I) and Zn(II), fitting of data for relative affinity constants, impact of acetonitrile on SEC experiments, stability of Cu(I) and Cu(I)-RING protein under ambient conditions.

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Splan, K.E., Choi, S.R., Claycomb, R.E. et al. Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions. J Biol Inorg Chem 28, 485–494 (2023). https://doi.org/10.1007/s00775-023-02002-4

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