The dynamics of metal binding to and transfer from metalloproteins involved in metal homeostasis are important for understanding cellular distribution of metal ions. The dicotyledonous plant Arabidopsis thaliana has two type 4 seed-specific metallothionein homologues, MT4a and MT4b, with likely roles in zinc(II) homeostasis. These two metallothioneins are 84% identical, with full conservation of all metal-binding cysteine and histidine residues. Yet, differences in their spatial and temporal expression patterns suggested divergence in their biological roles. To investigate whether biological functions are reflected in molecular properties, we compare aspects of zinc(II)-binding dynamics of full-length MT4a and MT4b, namely the pH dependence of zinc(II) binding and protein folding, and zinc(II) transfer to the chelator EDTA. UV–Vis and NMR spectroscopies as well as native electrospray ionisation mass spectrometry consistently showed that transfer from Zn 6 MT4a is considerably faster than from Zn 6 MT4b, with pseudo-first-order rate constants for the fastest observed step of k obs = 2.8 × 10 −4 s −1 (MT4b) and k obs = 7.5 × 10 −4 s −1 (MT4a) (5 µM protein, 500 µM EDTA, 25 mM Tris buffer, pH 7.33, 298 K). 2D heteronuclear NMR experiments allowed locating the most labile zinc(II) ions in domain II for both proteins. 3D homology models suggest that reactivity of this domain is governed by the local environment around the mononuclear Cys 2 His 2 site that is unique to type 4 MTs. Non-conservative amino acid substitutions in this region affect local electrostatics as well as whole-domain dynamics, with both effects rendering zinc(II) ions bound to MT4a more reactive in metal transfer reactions. Therefore, domain II of MT4a is well suited to rapidly release its bound zinc(II) ions, in broad agreement with a previously suggested role of MT4a in zinc(II) transport and delivery to other proteins.
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Acknowledgements We thank Prof. Peter Goldsbrough (Purdue University) for expression constructs for A. thaliana MT4a and MTb, the Royal Society (Olga Kennard Fellowship to CAB), the University of Warwick, and the Department of Chemistry (Chancellor’s Scholarship to HTI). We also thank Dr. Ralf Schmid (University of Leicester) for help with calculations of electrostatic potentials, and Dr. Esther Martin for helpful discussions regarding mass spectrometry. Support by Advantage West Midlands and the European Regional Development Fund (Birmingham Science City) is gratefully acknowledged.
© 2017, The Author(s).
Copyright 2019 Elsevier B.V., All rights reserved.
- Mass spectrometry
- Metal homeostasis
- Nuclear magnetic resonance
ASJC Scopus subject areas
- Inorganic Chemistry