Copper Binding to Rabbit Liver Metallothionein

Abstract

Circular dichroism and ultraviolet absorption spectral data have been used to probe the binding mechanism for formation and the structure of the copper(1)-thiolate binding clusters in rabbit liver metallothiionein during addition of Cu' to aqueous solutions of Zn,-metallothionein 2 and Cd,Zn,-metallothionein 2. Mammalian metallothionein binds metals in two binding sites, namely the a and P domains. Spectral data which probe the distribution of Cu(1) between the two binding domains clearly show that both the site of binding (a or p), and the structures of the specific metal-thiolate clusters formed, are dependent on temperature and on the nature of the starting protein (either Zn,-metallothionein or Cd,Zn,-metallothionein). CD spectra acquired during the addition of Cu' to Zn,-metallothionein show that Cu' replace the bound Zn(I1) in a domain-distributed manner with complete removal of the Zn(I1) after addition of 12 Cu'. Spectral and metal analyses prove that a series of Cu(1)-metallothionein species are formed by a non-cooperative metal-binding mechanism with a continuum of Cu(1) :metallothionein stoichionietries. Observation of a series of spectral saturation points signal the formation of distinct optically active Cu(1)-thiolate structures for the Cu,Zn,-metallothionein, Cu,,-metallothionein, and the Cu,,-metallothiionein species. These data very clearly show that for Cu(1) binding to Zn,-metallothionein, there are sleveral key Cu(1):metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu,,-metallothionein species are defined by bands located at 255(+) nm and 280(--) nm. Interpretation of the changes in the CD and ultraviolet absorption spectral data recorded between 3°C and 52°C as Cu' is added to Zn-metallothionein show that copper-thiolate cluster formation is strongly temperature dependent. These changes in spectral properties are interpreted in terms of kinetic versus thermodynamic control of the metal-binding pathways as Cu' binds to the protein. At low temperatures (3 "C and 10OC) the spectral data indicate a kinetically controlled mechanism whereby an activation barrier inhibits formation of ordered copper-thiolate structures until formation of Cu,,-metallothionein. At higher temperatures (>3OoC) the activation barrier is overcome, allowing formation of new Cu(1)-thiolate clusters with unique spectral properties, especially at the Cu,Zn,-metallothionein point. The CD spectra also show that a Cu,,-metallothionein species with a well-defined, three-dimensional structure forms at all temperatures, characterized by a band near 335 nm, indicating the presence of digonal Cu(1). Complicated CD spectral changes are observed when Cu' is added to Cd,Zn,-metallothionein. The spectral data are interpreted in terms of domain-distributed binding followed by rearrangement to form the domain-specific product. In the Cu,Cd,-metallothionein species, the Cu' are ultimately bound specifically to the p domain of the protein. This complex is characterised by the CD spectrum o...

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

“…Similar to our observations, Neurospora Cu-MT has negative CD at 290 nm and positive CD at 360 nm (32). Moreover, mixed Zn-Cu MTs have positive CD features at 360 nm and negative ones at 280 nm that depend on total metal and Cu∕Zn ratio (30). In MTs, the signals are proposed to arise from d 10 − d 10 interactions of adjacent metal ions when the metal-metal distances are relatively short (i.e., <2.8 Å) (33).…”

“…This conclusion is based on the highwavelength CD signals (around 350 nm) that match signals found in multi-metal-cluster MTs (30,31). It is possible that the folded Cu-cisPt-Atox1 complex is not monomeric but may to some degree resemble a recently reported Mo-Cu-Atx1 complex (47), formed when the copper-depleting drug tetrathiomolbydate was added to the yeast Cu chaperone, Atx1.…”

“…Specifically, the peak at 260 nm becomes more negative, a new negative shoulder at 300 nm arises, and a positive peak at 370 nm appears. These changes are reminiscent of CD signals of metallothioneins (MTs) (30,31), small Cys-rich peptides that bind many metals (up to 18) in distinct clusters involving bridging sulfurs. Similar to our observations, Neurospora Cu-MT has negative CD at 290 nm and positive CD at 360 nm (32).…”

Control of zinc transfer between thionein, metallothionein, and zinc proteins

“…At present, the precise structure of this species is unknown. Rearrangement of Zn 6 MT-2, perhaps with the formation of two Zn 3 clusters, might occur because the coordination of MT is quite flexible as demonstrated for copper MT, where an MT species containing one Cu 6 S 9 and one Cu 6 S 11 unit has been reported (28). MT exhibits high structural flexibility, and cadmium exchange inside the ␤-cluster and between the ␤-clusters of two MT molecules is fast (29).…”

Interaction between the Anticancer Drug Cisplatin and the Copper Chaperone Atox1 in Human Melanoma Cells

“…We recently demonstrated that CisPt can bind to Atox1 in vitro, both in the absence and the presence of Cu [10]. When Cu was pre-bound to Atox1, CisPt binding resulted in the appearance of circular dichroism (CD) signals above 300 nm, likely originating from metal-metal interactions [11][12][13]. Thus, CisPt binding to Cu-Atox1 resulted in a ternary CisPt-Cu-Atox1 complex.…”