title: Putting the pieces into place: Properties of intact zinc metallothionein 1A determined from interaction of its isolated domains with carbonic anhydrase.
abstract: Mammalian metallothioneins (MTs) bind up to seven Zn(2+) 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.
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7.
authors.forename: Tyler B J
authors.initials: TB
authors.lastname: Pinter
authors.affiliation: Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7 martin.stillman@uwo.ca.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stillman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: C530099:MT1A protein, human; D002104:Cadmium; D008668:Metallothionein; D002256:Carbonic Anhydrases; D015032:Zinc; D003545:Cysteine
keywords: binding affinity; competition; domain specificity; mass spectrometry; metallothionein; zinc