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.
Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase
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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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