Single-domain metallothioneins: Evidence of the onset of clustered metal binding domains in Zn-rhMT 1a

摘要

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.