Arsenic-metalation of triple-domain human metallothioneins: Support for the evolutionary advantage and interdomain metalation of multiple-metal-binding domains

Ngu, Thanh T.; Lee, Janice A.; Pinter, Tyler B. J.; Stillman, Martin J.

doi:10.1016/j.jinorgbio.2009.12.007

Cited by 14 publications

(10 citation statements)

References 51 publications

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“…While the fully metalated proteins have been studied in detail by numerous methods, there are few studies that specifically focus on the metal binding to the apoprotein. ESI-MS data for stepwise As 3+ binding showed that each As 3+ ion was bound to three cysteinyl thiolates with no clustering. ,, Noncooperative metalation has been established for Zn 2+ , Cd 2+ , − Bi 3+ , and As 3+ . ,,, Two recent papers , have discussed the binding of zinc to metallothionein and reported binding constants. The study by Namdarghanbari et al using the zinc sensor FluoZin-3 reported that all the stability constants were approximately 10 11 and that there was no evidence of a stability constant as low as ∼10 8 that had been previously reported by Krezel and Maret…”

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

2013

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“…There is also a 20% decrease in the intensity of the 3+ relative to the 4+ charge state when comparing apo-β-rhMT to Zn 3 -β-rhMT. We should note at this point that several ESI-MS studies, specifically the kinetic analysis of arsenic metalation, 2,25,27,28 provide strong evidence that the ionization potential of MT, and its isolated domains, are similar for all metalation states (from apo-MT to fully metalated MT), and, therefore, the deconvoluted spectra are good approximations of the actual metal ion distribution in solution. Taken together, these results suggest that the addition of the first two metal ions stabilize a slightly more open conformation of apo-β-rhMT.…”

Section: ■ Resultsmentioning

confidence: 98%

“…Finally, the recent kinetic studies of As 3+ metalation of hMT1 demonstrated conclusively that the affinity of MT for As 3+ is directly dependent upon the number of available metal binding sites, ,, with the specific rate constants declining linearly as the number of As 3+ bound increased forming a chain of cys-coordinated 6(AsS 3 ). For a cooperative mechanism, the specific rate constants would increase for each successive As 3+ bound.…”

Section: Discussionmentioning

confidence: 94%

“…In a noncooperative mechanism of metalation, the binding affinity of MT for metal ions is directly dependent upon the number of available metal-binding sites, and the binding affinity decreases for each successive metalation event in the normal and expected pattern of coordination chemistry. , This means that in the absence of excess metal ions the last metal binding site might not be occupied, leading to a partially metalated species. The kinetic data of Ngu et al provided clear evidence for this effect ,,, because the series of specific rate constants determined from the time-dependent data diminished for each successive metal bound up the sixth As 3+ .…”

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“…3,26 This means that in the absence of excess metal ions the last metal binding site might not be occupied, leading to a partially metalated species. The kinetic data of Ngu et al provided clear evidence for this effect 2,25,27,28 because the series of specific rate constants determined from the time-dependent data diminished for each successive metal bound up the sixth As 3+ .…”

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Noncooperative Metalation of Metallothionein 1a and Its Isolated Domains with Zinc

2012

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Mammalian metallothioneins (MTs) are a family of small cysteine-rich proteins capable of binding 7 Zn(2+) or Cd(2+) ions into two distinct domains: an N-terminal β-domain that binds 3 Zn(2+) or Cd(2+) and a C-terminal α-domain that binds 4 Zn(2+) or Cd(2+). MT has been implicated in a number of physiological functions, including metal ion homeostasis, toxic metal detoxification, and as a protective agent against oxidative stress. Conventionally, MT has been understood to coordinate metal ions in a cooperative fashion. Under this mechanism of metalation, the only species of biological relevance would be the metal-free (apo-) form of the protein and the fully metalated (holo-) form of the protein. However, an increasing body of evidence suggests that metalation occurs in a noncooperative manner. If this latter mechanism is correct, then partially metalated forms of the protein will be stable and able to take part in cellular chemistry. We report in this paper conclusive evidence that shows that biologically essential zinc binds to MT in a noncooperative manner. In addition, we report for the first time the stability of a Zn(5)-MT species. The implications of these findings are discussed in terms of the mechanism of metalation.

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Metallation of SeaweedFucus vesiculosusMetallothionein: As³⁺and Cd²⁺binding

Ngu

Stillman

2011

Handbook of Marine Macroalgae

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Arsenic-metalation of triple-domain human metallothioneins: Support for the evolutionary advantage and interdomain metalation of multiple-metal-binding domains

Cited by 14 publications

References 51 publications

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

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

Noncooperative Metalation of Metallothionein 1a and Its Isolated Domains with Zinc

Metallation of SeaweedFucus vesiculosusMetallothionein: As³⁺and Cd²⁺binding

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Arsenic-metalation of triple-domain human metallothioneins: Support for the evolutionary advantage and interdomain metalation of multiple-metal-binding domains

Cited by 14 publications

References 51 publications

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

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

Noncooperative Metalation of Metallothionein 1a and Its Isolated Domains with Zinc

Metallation of SeaweedFucus vesiculosusMetallothionein: As3+and Cd2+binding

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Metallation of SeaweedFucus vesiculosusMetallothionein: As³⁺and Cd²⁺binding