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.

Selective cysteine modification of metal‐free human metallothionein 1a and its isolated domain fragments: Solution structural properties revealed via ESI‐MS

“…The key to these semi‐quantitative data is the assumption that apo‐MTs and those with different numbers of metals coordinated exhibit very similar ionization efficiencies; this assumption has been supported by the literature, especially for metalation of metallothioneins . While it is true that the different MT‐isoforms will have slightly varying ionization efficiencies, when analyzing modifications of the same isoform, the behavior of all species in solution has been demonstrated to be predictable and the metal‐binding parameters determined align closely with those determined by other methods …”

“…32 ESI-mass spectral data have been used to determine structural properties of apo-MT 25 and MT partially metalated with As 31 24 and Cd 21 . 28,33 ESI-MS data can also be used to determine quantitative biochemical parameters as recently demonstrated in the determination of arsenic metalation kinetics, 34 cadmium and zinc equilibrium, and kinetic constants 28,35,36 and copper binding affinity. 37 The key to these semi-quantitative data is the assumption that apo-MTs and those with different numbers of metals coordinated exhibit very similar ionization efficiencies; this assumption has been supported by the literature, especially for metalation of metallothioneins.…”

“…38 While it is true that the different MTisoforms will have slightly varying ionization efficiencies, 39 when analyzing modifications of the same isoform, the behavior of all species in solution has been demonstrated to be predictable and the metalbinding parameters determined align closely with those determined by other methods. 36,40,41 Previous studies using cysteine modification agents to probe the solution structure and metal binding properties of MTs have focused on one modifier at a time, leading us to question whether there was a bias based on the size and relative solvation properties of the modifying molecule.…”

Residue Modification and Mass Spectrometry for the Investigation of Structural and Metalation Properties of Metallothionein and Cysteine-Rich Proteins

“…Domain specificity is a concept that has long been investigated in the case of metallothioneins with its two-domain structure consisting of a smaller, 9 cysteine β-domain and a larger 11-cysteine α-domain [94,95,96,97,98]. Domain specificity has been primarily investigated by NMR spectroscopy [95] and ESI-MS [98,99] due to the ability of both techniques to identify the domain in which the metal is bound.…”

Proteomic High Affinity Zn2+ Trafficking: Where Does Metallothionein Fit in?

“…Subsequently, an elegant series of experiments by Stillman and his students, also using ESI, strongly supported the stepwise formation of Zn 7 -MT from Zn 2+ and apo-MT [32,43,44]. In particular, when apo-MT, apo-CA, and various concentrations of Zn 2+ were mixed, the metal ion distributed itself among Zn-CA and Zn n -MT ( n = 1–7) such that equilibrium constants for the binding of each Zn 2+ to MT could be determined as described above [32].…”