Mammalian metallothioneins (MTs) bind up to seven Zn2+ using a large number of cysteine residues relative to their small size and can act as zinc-chaperones. In metal-saturated Zn7–MTs, the seven zinc ions are co-ordinated tetrahedrally into two distinct clusters separated by a linker; the N-terminal β-domain [(Zn3Cys9)3−] and C-terminal α-domain [(Zn4Cys11)3−]. We report on the competitive zinc metalation of apo-carbonic anhydrase [CA; metal-free CA (apo-CA)] in the presence of apo-metallothionein 1A domain fragments to identify domain specific determinants of zinc binding and zinc donation in the intact two-domain Znn–βαMT1A (human metallothionein 1A isoform; n=0–7). The apo-CA is shown to compete effectively only with Zn2–3–βMT and Zn4–αMT. Detailed modelling of the ESI mass spectral data have revealed the zinc-binding affinities of each of the zinc-binding sites in the two isolated fragments. The three calculated equilibrium zinc affinities [log(KF)] of the isolated β-domain were: 12.2, 11.7 and 11.4 and the four isolated α-domain affinities were: 13.5, 13.2, 12.7 and 12.6. These data provide guidance in identification of the location of the strongest-bound and weakest-bound zinc in the intact two-domain Zn7βαMT. The β-domain has the weakest zinc-binding site and this is where zinc ions are donated from in the Zn7–βαMT. The α-domain with the highest affinity binds the first zinc, which we propose leads to an unscrambling of the cysteine ligands from the apo-peptide bundle. We propose that stabilization of the intact Zn6–MT and Zn7–MT, relative to that of the sum of the separated fragments, is due to the availability of additional cysteine ligand orientations (through interdomain interactions) to support the clustered structures.
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Research Article|
October 16 2015
Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase
Tyler B.J. Pinter;
Tyler B.J. Pinter
*Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7
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Martin J. Stillman
Martin J. Stillman
1
*Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7
1To whom correspondence should be addressed (email martin.stillman@uwo.ca).
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Publisher: Portland Press Ltd
Received:
June 10 2015
Revision Received:
August 03 2015
Accepted:
August 20 2015
Accepted Manuscript online:
August 20 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2015 Authors; published by Portland Press Limited
2015
Biochem J (2015) 471 (3): 347–356.
Article history
Received:
June 10 2015
Revision Received:
August 03 2015
Accepted:
August 20 2015
Accepted Manuscript online:
August 20 2015
Citation
Tyler B.J. Pinter, Martin J. Stillman; Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase. Biochem J 1 November 2015; 471 (3): 347–356. doi: https://doi.org/10.1042/BJ20150676
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