Single-Domain Metallothioneins: Evidence of the Onset of Clustered Metal Binding Domains in Zn-rhMT 1a

Abstract

Mammalian metallothioneins bind up to seven Zn(2+) ions in two distinct domains: an N-terminal β-domain that binds three Zn(2+) ions and a C-terminal α-domain that binds four Zn(2+) ions. Domain specificity has been invoked in the metalation mechanism with cluster formation and bridging of the 20 Cys residues taking place prior to saturation with seven Zn(2+) ions. We report a novel experiment that examines Zn(2+) metalation by exploiting the expected decrease in K(F) at the onset of clustering using electrospray ionization mass spectrometry (ESI-MS). During the titration with Zn(2+), the ESI-MS data show that several metalated species coexist until the fully saturated proteins are formed. The relative Zn binding affinities of the seven total sites in the α- and β-fragments were determined through direct competition for added Zn(2+). The K(F) values for each Zn(2+) are expected to decrease as a function of the remaining available sites and the onset of clustering. Analysis shows that Zn(2+) binds to β-rhMT with a greater affinity than α-rhMT. The incremental distribution of Zn(2+) between the competing fragments and apo-βα-rhMT (essentially three and four sites competing with seven sites) identifies the exact point at which clustering begins in the full protein. Analysis of the speciation data shows that Zn(5)-MT forms before clustering begins. This means that all 20 Cys residues of apo-βα-rhMT are bound terminally to Zn(2+) as [Zn(Cys)(4)](2-) units before clustering begins; there is no domain preference in this first metalation stage. Preferential binding of Zn(2+) to β- and α-rhMT at the point where βα-rhMT must form clusters is caused by a significant decrease in the affinity of βα-rhMT for further Zn(2+). The single-domain Zn(5)-rhMT, in which there are no exposed cysteine sulfurs, is a key component of the metalation pathway because the lower affinities of the two clustered Zn(2+) ions allow donation to apoenzymes.

Metalation Kinetics of the Human α‐Metallothionein 1a Fragment Is Dependent on the Fluxional Structure of the apo‐Protein

“…This process has been recently discussed for Zn-MT. [43] The structural determinants for this process are unknown. However, elucidation of The metal-induced folding of apo-a-MT is driven by the stepwise formation of the metal-thiolate cluster within the core of the domain.…”

“…It is likely that in the globular conformation, the cysteine residues are prearranged to the correct orientation for cluster formation and therefore, this final rearrangement step is fast. The denatured protein may be able to bind the first two metals more quickly in a bead-like structure previously described, [43] but once bridging cysteines are required for the cluster structure, the necessary structural rearrangement is much slower. Although one might expect the open, string-like conformation to metalate more rapidly due to the increased accessibility of cysteine residues compared to the globular state, the results of the metalation experiment with Cd II show unambiguously that the opposite is true.…”

Advances in the molecular understanding of biological zinc transport

“…233 Finally, titrations of human apo-MT-1a with various metal ions including Zn 2+ using a stopped-flow approach showed that this MT also binds these metal ions non-cooperatively, as yet again the observed metallo-species did not indicate a preference for complete clusters. 235,236 Since MTs can be populated by cadmium in vivo, 177,178 it is of interest to study the replacement of Zn 2+ by Cd 2+ . Spectrophotometric analysis of the kinetics of Cd 2+ /Zn 2+ exchange in rabbit MT-2 as well as the isolated a domain revealed that metal exchange occurs via an associative mechanism, with the incoming Cd 2+ initially binding to the fully metallated MT, followed by exchange with a bound Zn 2+ .…”

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

“…26,28,47,49 In the formation of metal-saturated final products, the MTs pass through partially-metalated 56 Assuming that the metalation reactions follow a purely domain-specific mechanism, it is likely that the separated domains would enhance this feature since interdomain interactions that would result in property differences between the separated domains and intact protein are reduced. 53 We will return to discuss evidence for interdomain interactions below.…”