Cu+ distribution in metallothionein fragments

Salgado, Maria T.; Stillman, Martin J.

doi:10.1016/j.bbrc.2004.03.183

Cited by 37 publications

(34 citation statements)

References 35 publications

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“…41,46 Previous metalation experiments, such as copper luminescence, 28,47 provide only an averaged measure of metalation, not the exact speciation over all sites. The discriminating capacity of ESI-MS provides simultaneous information about all species present and their respective dynamic metalation states.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

2013

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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.

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Section: ■ Results and Discussionmentioning

confidence: 99%

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

2013

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“…[24,29,38] Despite this extensive literature, the details of the stepwise metalation mechanism by which the 7 Zn II or Cd II ions bind to the apo-MT is poorly understood. However, it is clear that the metalation reaction follows a distinct pathway because in the case of both Cu I [9,40] and As III [34,37] kinetic data have been reported that are metal-status dependent. By this we mean that the time-dependent data recorded during these metalation reactions are sensitive to the metal loading from the apoprotein to full saturation.…”

Section: Molecular Dynamics (Md) Modelsmentioning

confidence: 93%

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

Irvine

Duncan

Gullons

et al. 2014

Chemistry A European J

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Mammalian metallothioneins are cysteine rich metal-binding proteins comprising, when fully metalated, two metal-binding domains: the α-domain binds with M4(II)S(Cys)11 stoichiometry and the β domain binds as M3(II)S(Cys)9 stoichiometry. While the fully metalated species have been widely studied, the metalation of the apoprotein is poorly understood. Key to a description of the metalation pathway(s) is the initial conformation of the apoprotein and the arrangement of the metal-coordinating cysteines prior to metalation. We report the effect of the ill-defined, globular structure of apoMT on metalation rates. In order to overcome the experimental limitations inherent in structural determinations of a fluxional protein we used a detailed analysis of the apo-α-metallothionein conformation based on the differential rate of cysteine modification with benzoquinone. The ESI mass spectral data show the presence of two distinct conformational families: one a folded conformational family at neutral pH and a second an unfolded family of conformations at low pH. The Cd(II) metalation properties of these two conformationally distinct families were studied using stopped-flow kinetics. Surprisingly, the unfolded apoprotein metalated significantly slower than the folded apoprotein, a result interpreted as being due to the longer time required to fold into the cluster structure when the fourth Cd(II) binds. These results provide the first evidence for the role of the structure of the apo-αMT in the metalation reaction pathways and show that cysteine modification coupled with ESI-MS can be used to probe structure in cysteine-rich proteins.

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“…In other words, the divalent metals Cd 2+ and Zn 2+ coordinate four metals in the a domain and three metals in the b domain because this is the maximum number that can be accommodated by the thiolate ligands imposed by the limitations of tetrahedral coordination. However, Cu + , Ag + and Hg 2+ are capable of adopting tetrahedral, trigonal and digonal coordination geometries, and as a result, both the a and b domains can achieve domain occupancies of four, six or nine metal ions depending on the extent of metal loading to the protein [37][38][39][40]. Even in the high metal occupancy species, the polypeptide backbone wraps around the cluster into a tightly folded conformation as determined by the generation of a CD spectrum, which suggests that all of the metallation species may have a role in the function of the protein.…”

Section: Metallation Of Apo-mtmentioning

confidence: 99%