Domain Selection in Metallothionein 1A: Affinity-Controlled Mechanisms of Zinc Binding and Cadmium Exchange

Abstract

Mammalian metallothioneins (MTs) are small, metal binding proteins implicated in cellular metal ion homeostasis and heavy metal detoxification. Divalent, metal-saturated MTs form two distinct domains; the N-terminal β domain binds three metals using nine Cys residues, and the C-terminal α domain binds four metals with 11 Cys residues. Domain selection during zinc binding and cadmium exchange to human MT1A was examined using a series of competition reactions with mixtures of the isolated domain fragments. These experiments were conducted at two biologically significant pH conditions where MTs exist in vivo. Neither zinc binding nor cadmium exchange showed any significant degree of specificity or selectivity based on detailed analysis of electrospray ionization mass spectrometric and circular dichroic data. Under acidic conditions, zinc binding and cadmium exchange showed slight α domain selectivity because of the increased preference for cooperative clustering of the α domain. Modeling of the reactions showed that at both physiological (7.4) and acidic (5.8) pHs, zinc binding and cadmium exchanges occur essentially randomly between the two fragments. The metal binding affinity distributions between the domain fragments are comingled and not significantly separated as required for a domain specific mechanism. The models show rather that the order of the binding events follows the order of the binding affinities that are distributed across both domains and that this can be considered quantitatively by the KF(Cd)/KF(Zn) binding constant ratio for each metal bound.

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

“…Although some isoform-specific differences likely exist, MT-1a cooperatively forms cadmium clusters in the α-domain and this has been reported in both the full-length protein and the isolated α-domain fragment [67,89]. In competition experiments, a slight preference for α-domain metalation is observed that is exaggerated at lower pH [98]. Thus at least for the MT-1/2 isoforms cadmium metalation is α-domain specific, a phenomenon that becomes more apparent at lower pH.…”

“…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.…”

“…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. This has led to reports of certain isoforms and domains having specific metal binding properties, resulting in some isoforms and domains labeled as being metal-specific [97,100,101].…”

“…Domain specificity presents a larger challenge, since RNA analysis cannot give information about intra-protein selection in response to metal stimuli. To address these questions, isolated domain competition experiments can be performed, although the elimination of the inter-domain linker raises questions about the strength of the chelate-effect of the entire protein and the barrier to cross domain metal exchange introduced by the separation of these domains in solution [98]. …”

“…The mammalian MT family is considerably less diverse than amongst other biological strata [106]. Interestingly, ionization efficiencies vary widely between the similar mammalian MT isoforms, and even between the two domains of MT1a which require external concentration verification in competition and affinity experiments [32,98,107]. …”