Effect of cysteine replacements at positions 13 and 50 on metallothionein structure

Cismowski, Mary J.; Huang, P. C.

doi:10.1021/bi00240a036

Cited by 45 publications

(29 citation statements)

References 25 publications

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“…5 E) and the complete lack of any correspondence of their CD spectrum with the calculated difference between the CD spectra of the Cd,-a,-fragment and the parent holoprotein suggest that the Cd-thiolate cluster in the Cd,-P-fragments has undergone a major transformation which we ascribe to the loss of Cys38 from the cluster. A similar large chiroptical change of a Cd-thiolate cluster has been reported earlier for recombinant Chinese hamster MT in which a metal-binding cysteine residue (CysSO) was replaced by tyrosine (Cismowski and Huang, 1991). Such large effects are believed to be the result of changes in the arrangement of the chromophoric thiolate ligands within the Cd-thiolate clusters and are thus a reflection of cluster geometry (Willner et al, 1992).…”

Section: Resultssupporting

confidence: 65%

Separation and Characterization of the Metal‐Thiolate‐Cluster Domains of Recombinant Sea Urchin Metallothionein

Wang

Heß

Hunziker

et al. 1996

European Journal of Biochemistry

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Partial metal depletion and "Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrutus purpurutus (Cd,-MTA) binds its metal ions in a four-metal (Cd,Cys,,) and a three-metal (Cd,Cys,) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E. A., Zerbe, O., Hess, D., Hunziker, P. E., Vasak, M. & Kagi, J. H. R. (1995) Biochemistry 34, 7460-74671. This partitioning has now been confirmed by bisecting native Cd,-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37-39 residues with four Cd ions and a set of C-terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal-bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N-terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd,-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster.Keywords: recombinant sea urchin metallothionein ; limited proteolysis; mass spectroscopy ; circular dichroism.Metallothioneins (MT) are small proteins with a high cysteine and d'" metal ion content (e.g. Znz+, Cd2+, Hg2 ' and Cu+).They are ubiquitous in the animal kingdom and are thought to play a central role in cellular Zn and Cu metabolism and in the protection from the toxic elements Cd and Hg (Kagi, 1991; Roesijadi, 1993). The metal ions are bound to the cysteine side chains and "'Cd-NMR studies of MT whose binding sites were fully occupied by "'Cd indicated that both in mammalian and in crustacean MT the metal complexes are joined to form two distinct metal-thiolate clusters (Otvos and Armitage, 1980;Otvos et al., 1982;Frey et al., 1985). This structure was subsequently confirmed by two-dimensional NMR and by X-ray crystallographic studies (Wuthrich, 1991 ;Robbins et al., 1991). Mammalian MT contain a M:+Cys,, and a M,2+CysI, cluster located in the interior of the essentially globular N-terminal and C-termina...

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Section: Resultssupporting

confidence: 65%

Separation and Characterization of the Metal‐Thiolate‐Cluster Domains of Recombinant Sea Urchin Metallothionein

Wang

Heß

Hunziker

et al. 1996

European Journal of Biochemistry

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“…We have suggested that this free thiol might be more reactive toward disulfides such as GSSG (4). Herein, we have used Ellman's reagent to investigate the redox behavior of MT, a reaction that has been studied earlier in some detail (13,14,34). Under the conditions of our experiments, the kinetic phase affected by ATP corresponds to the faster of the two phases ascribed by others to the reaction of the ␣-domain of MT (35).…”

Section: Discussionmentioning

confidence: 94%

The ATP–metallothionein complex

Jiang

Maret

Vallée

1998

Proc. Natl. Acad. Sci. U.S.A.

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We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (K d ‫؍‬ 176 ؎ 33 M, pH 7.4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol-disulfide interchange with Ellman's reagent [5,5-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di-or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two critical cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding constant and the cellular concentration of ATP indicate that in the cell MT could be essentially saturated with ATP at low concentrations of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.Seven zinc atoms bound to 20 cysteine sulfur atoms form the two metal clusters of metallothionein (MT) and constitute a reservoir for storage and distribution of zinc (1). The cluster structure forms a network of zinc-sulfur interactions that have no precedent in the nonliving world. The protein envelops the zinc atoms, each of which is bound to four thiolate ligands, in a manner that effectively shields them from the environment. Ligand exchange and redox reactions of the cysteine sulfur donor atoms are responsible for the kinetic lability of zinc in the thermodynamically stable sites of MT (2). The cluster unit works by a mechanism that allows the cysteine sulfur ligands to zinc to be oxidized, allowing an oxidoreductive mechanism to modulate affinity of the otherwise inert zinc atom (3). We have shown further that the interactions of MT with glutathione (GSH) and glutathione disulfide (GSSG) (4) or other oxidizing agents (5) control the state of zinc in MT in vitro, suggesting that the zinc content of MT is a function of the cellular redox state. Aside from GSH (6), the only other biological ligand known to bind to MT, until now, is phosphate that appears to be required for the formation of stable MT crystals (7). It was found to be localized between the two protein domains bound to the carbonyl group of Cys-19 and to the -amino group of Lys-31, which forms the only interdomain hydrogen bonds with the carbonyl groups of Cys-19 and Cys-21 (7). It has also been found to bind to dimeric Cd-MT with relativ...

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“…The near-UV CD might be even better owing to its sensitivity to small perturbations of the environments of the aromatic side chains. Indeed, some researchers have used the near-UV CD for this purpose (Cismowski and Huang, 1991;Lin et at., 1992;van der Goot et al, 1993a,b;Patti et al, 1993), but a larger number have used the far-UV CD only. Ideally, both the near-and far-UV CD spectra should be used for comparing altered and wildtype proteins.…”

Section: A Structural Analysis Of Recombinant Native Proteins and Thmentioning

confidence: 99%

Aromatic and Cystine Side-Chain Circular Dichroism in Proteins

Woody

Dunker

1996

Circular Dichroism and the Conformational Analysis of Biomolecules

188

192

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Effect of cysteine replacements at positions 13 and 50 on metallothionein structure

Cited by 45 publications

References 25 publications

Separation and Characterization of the Metal‐Thiolate‐Cluster Domains of Recombinant Sea Urchin Metallothionein

Separation and Characterization of the Metal‐Thiolate‐Cluster Domains of Recombinant Sea Urchin Metallothionein

The ATP–metallothionein complex

Aromatic and Cystine Side-Chain Circular Dichroism in Proteins

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