Imam, Hasan T. and Blindauer, Claudia A. (2018) Differential reactivity of closely related zinc(II)-binding metallothioneins from the plant Arabidopsis thaliana. Journal of Biological Inorganic Chemistry, 23 (1). pp. 137-154. doi:10.1007/s00775-017-1516-6 ISSN 0949-8257.

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Abstract

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 Zn6MT4a is considerably faster than from Zn6MT4b, 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 Cys2His2 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.

Item Type:	Journal Article
Subjects:	Q Science > QD Chemistry
Divisions:	Faculty of Science, Engineering and Medicine > Science > Chemistry
Library of Congress Subject Headings (LCSH):	Zinc compounds, Zinc -- Physiological effect, Metallothionein, Zinc proteins, Nuclear magnetic resonance, Mass spectrometry, Arabidopsis thaliana
Journal or Publication Title:	Journal of Biological Inorganic Chemistry
Publisher:	Springer
ISSN:	0949-8257
Official Date:	January 2018
Dates:	Date Event January 2018 Published 7 December 2017 Available 12 November 2017 Accepted
Volume:	23
Number:	1
Page Range:	pp. 137-154
DOI:	10.1007/s00775-017-1516-6
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access (Creative Commons)
Copyright Holders:	The Author(s) 2017
Date of first compliant deposit:	13 March 2018
Date of first compliant Open Access:	13 March 2018
RIOXX Funder/Project Grant:	Project/Grant ID RIOXX Funder Name Funder ID Olga Kennard Fellowship Royal Society http://dx.doi.org/10.13039/501100000288 Chancellor’s International Scholarship University of Warwick http://dx.doi.org/10.13039/501100000741 UNSPECIFIED Advantage West Midlands UNSPECIFIED UNSPECIFIED European Regional Development Fund http://dx.doi.org/10.13039/501100008530

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