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

摘要

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 Zn-7-MTs, the seven zinc ions are co-ordinated tetrahedrally into two distinct clusters separated by a linker; the N-terminal beta-domain [(Zn(3)Cys(9))(3-)] and C-terminal alpha-domain [(Zn(4)Cys(11))(3-)]. We report on the competitive zinc metalation of apo-carbonic anhydrase [CA; metal-free CA (apo-CA)] in the presence of apometallothionein 1A domain fragments to identify domain specific determinants of zinc binding and zinc donation in the intact two-domain Zn-n-beta alpha MT1A (human metallothionein 1A isoform; n=0-7). The apo-CA is shown to compete effectively only with Zn2-3-beta MT and Zn-4-a alpha MT. 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(K-F)] of the isolated beta-domain were: 12.2, 11.7 and 11.4 and the four isolated alpha-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 Zn-7 beta alpha MT. The beta-domain has the weakest zinc-binding site and this is where zinc ions are donated from in the Zn-7-beta alpha MT. The alpha-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 Zn-6-MT and Zn-7-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.