From the University of Maryland Center for
Environmental Science, Chesapeake Biological Laboratory, Solomons,
Maryland 20688 and ¶ Institute of Biochemistry, University of
Zürich, Winterthurerstrasse 190, CH-8057 Zürich,
Switzerland
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ABSTRACT |
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The ability of metallothionein (MT) to modulate
DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc
transcription factor, was investigated using metal-bound and
metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein
inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding
by zinc-deficient apo-TTK. "Free" zinc at low concentrations was as
effective as Zn-MT in restoring DNA binding by apopeptide but was
inhibitory at concentrations equal to zinc bound to 2 mol eq and higher
of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the
peptide for its DNA binding site. This effect was reversed by
incubation with Zn-MT. The circular dichroic spectra of the TTK peptide
indicated that zinc removal resulted in loss of -helical structures,
which are sites of DNA contact points. Reconstitution with cadmium
resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide
but not recovery of
-helical structures. Incubation of Cd-TTK with
Zn-MT restored the secondary structure expected for zinc-bound TTK. The
ability of Zn-MT and thionein to restore or inhibit DNA-binding by TTK
was associated with effects on the metallation status of the peptide
and related alterations in its secondary structure.
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INTRODUCTION |
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The low molecular weight metal-binding protein metallothionein (MT)1 is proposed to have functions in metal ion regulation and detoxification (1) and as a scavenger of free radicals (2). Roles for MT in dynamic intermolecular metal exchange reactions are consistent with the high thermodynamic stability (3) and high kinetic lability of its metal binding sites (4, 5). Metal exchange can also be facilitated by cellular redox couples such as GSH and GSSG (6-9). An early focus on enzyme activation by Zn-MT implicated MT as a zinc donor to apometallic forms of metalloenzymes such as aldolase, thermolysin, and carbonic anhydrase (10) and pyridoxal kinase (11). Recent studies on the effect of MTs on interactions between zinc transcription factors and their cognate DNAs implicate MTs in gene regulation. Incubation of metal-free thionein with CCHH zinc finger transcription factors Sp1 (12) and TFIIIA (13) inhibits DNA binding, suggesting zinc abstraction by thionein as a mechanism for regulating gene expression. Reciprocal zinc exchange is suggested as a mechanism for regulating gene expression in a study in which Zn-MT and thionein could activate or inhibit DNA binding by the estrogen receptor (14), a CCCC zinc finger protein.
Zinc transcription factors appear uniquely qualified as potential
partner molecules in metal exchange reactions with MT. Reported stability constants for zinc binding by zinc finger peptides and proteins are in a similar range, i.e. within 1 or 2 orders
of magnitude, as that of MTs (15-17). Additionally, it appears that the kinetics for zinc exchange in these structures are also relatively rapid (18). Direct examination of the metal transfer process has shown
that zinc can be transferred from an -domain peptide of human MT-2
(
-hMT-2) to a peptide derived from the third finger of Sp1 (16) and
from rabbit Zn-MT-2 to the Zn-transcription factor GAL4 (19). The rate
constant for zinc exchange for GAL4 is rapid and similar to that of MTs
(18, 19). Although the functional consequences of the transfers were
not addressed in these studies; considered together with the preceding
examples, it appears that MT can modulate DNA binding of zinc
transcription factors by regulating the availability of zinc.
The notion that Zn-MT can modulate intracellular zinc availability can
also be extended to interactions of zinc metalloproteins with toxic
metals whose toxicity is thought to be due, in part, to their
displacement of zinc (20). This condition is considered to be
reversible through reactions with Zn-MT, which has the potential to
abstract a toxic metal and donate an essential metal. Inhibition of
carboxypeptidase activity by cadmium, for example, can be reversed by
incubation with Zn-MT (21). This model for participation of MT in metal
detoxification has been designated as a "rescue" or "repair"
function that confers on Zn-MT an active, as opposed to a passive, role
in the protective response to metals (21, 22). It is known that cadmium
binding to some zinc finger proteins reduces the affinity of the latter
for their DNA-binding sites (23) and that metal-metal exchange can
occur between Zn-MT and zinc transcription factors bound to other
metals, e.g. between nickel-bound Sp1-3 zinc finger peptide
and zinc-bound -hMT-2 peptide (16). These observations raise the
possibility that Zn-MT can restore DNA binding activity to zinc
transcription factors through metal-metal exchange reactions.
Our study addressed the ability of thionein and Zn-MT to modulate
sequence-specific DNA binding by a two-zinc finger peptide (24) of
Tramtrack (TTK) (25, 26) prepared in different metallation states
(i.e. as zinc-bound, apo-TTK, and cadmium-bound states). TTK
is a classical CCHH transcription factor (27) that suppresses genes
involved in regulating development and differentiation in Drosophila (28). The zinc atom in a single zinc finger of
TTK is tetrahedrally coordinated in a motif that includes an
antiparallel -sheet containing two cysteine ligands and a short
-helix containing two histidine ligands (29). The amino acid to base
contacts reside in the
-helix of the zinc finger motif (29).
Experiments tested the feasibility of 1) reciprocal zinc exchange between TTK and MT in modulating the DNA binding by TTK and 2) cadmium-zinc exchange between Cd-TTK and Zn-MT as a mechanism for cadmium detoxification. Band shift gels were used to assess the effect of various metallation states of MT and the TTK peptide on sequence-specific DNA binding of the latter. Changes in the secondary structure of the TTK peptide and interactions between MT and TTK were monitored by measuring CD. Stepwise substitution of cadmium for zinc in TTK was followed by electronic absorption spectroscopy. The results showed that the peptide structure was dependent on its metallation state and implicated the Zn-MT-thionein conjugate pair in modulation of DNA-binding activity and detoxification of cadmium.
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EXPERIMENTAL PROCEDURES |
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Expression of TTK Peptide and Preparation of Metallated and
Nonmetallated Forms--
The TTK peptide 911zf was cloned in pET11a
(24) and expressed in Escherichia coli strain BL21(DE3)
(Novagen, Inc). The peptide was extracted from inclusion bodies in the
zinc-renatured form basically as described in Fairall et al.
(24) and purified by fast protein liquid chromatography (Amersham
Pharmacia Biotech) with HiTrap Heparin affinity columns (Amersham
Pharmacia Biotech) equilibrated in either 20 mM MES, pH
6.0, 1 mM DTT, 50 µM ZnSO4 or 20 mM Tris-HCl, pH 7.5, 2 mM DTT, 0.1 M NaCl, 50 µM ZnSO4. The TTK
peptide was eluted with a linear NaCl gradient with 1 M
NaCl in the respective limit buffer. Purification was monitored by
SDS-polyacrylamide gel electrophoresis using 4-20% or 10-20% gradient gels (Bio-Rad). The purified peptide was dialyzed against 20 mM Tris-HCl, pH 7.5, 2 mM DTT, 0.1 M NaCl, 50 µM ZnSO4 and stored at
80 °C following the addition of glycerol to a 10% final concentration. Prior to the addition of glycerol, an aliquot of the
peptide was dialyzed against 5 mM ammonium acetate and
analyzed by ESI-MS with a SCIEX APIII+ triple quadrupole instrument
under both acidic (sample diluted 1:1 with 50% methanol, 1% formic
acid) and neutral (5 mM ammonium acetate) conditions for
confirmation of the expected masses of the apo form and zinc-bound
form, respectively. For use in band shift gels, TTK peptide was
dialyzed against 20 mM Tris-HCl, pH 7.5, 0.1 M
NaCl, 1 mM mercaptoethanol to remove unbound zinc or
processed as described below for preparation of the apo and cadmium
forms.
Preparation of Apo- and Zn-MT-- Cadmium-induced rabbit MT-2 purified by gel permeation and ion exchange chromatography (32) was used for preparation of apo- and Zn-MT (30). ESI-MS under acidic conditions showed that this MT-2 preparation consisted of roughly equivalent amounts of MT-2a and MT-2c (nomenclature of Ref. 33).
MT concentrations were estimated from absorbance at 220 nm using a molar extinction coefficient of 48,200 MBand Shift Gels-- Band shift gels were conducted basically as described by Fairall et al. (24), substituting 10% polyacrylamide gels for agarose and omitting EDTA from the electrophoresis buffer and zinc from the peptide-DNA binding buffer. The electrophoresis buffer was 45 mM Tris borate; the binding buffer consisted of 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 40 mM NaCl, 0.1% Nonidet P-40, 10 µg/ml poly[d(I-C)], and 10% glycerol in a total reaction volume of 20 µl. Binding site DNA was added to each reaction to a final concentration of 10 nM.
Oligonucleotides for the binding site DNA encoded the 18-base pair oligonucleotide,
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Absorption and CD Spectroscopy--
Absorption spectra were
recorded on a Cary 3 spectrophotometer in a 1-cm quartz cuvette with
the sample in 10 mM Tris-HCl, pH 7.4, 0.1 M
NaCl. CD measurements were performed on a Jasco (model J-715)
spectropolarimeter using a 0.1-cm cylindrical quartz cuvette. The CD
data were expressed as mean residue molar ellipticity [] (degrees
dmol
1 cm2) or as ellipticity (millidegrees)
as appropriate. Secondary structure analysis of the CD spectra utilized
the method of Yang (35) as implemented in the protein secondary
structure estimation program provided by the manufacturer (Jasco). The
program estimates the content of
-helix,
-sheet,
-turn, and
unordered structures by a least-square method. The CD spectra for the
zinc-bound TTK and Cd-TTK were determined in 2.5 mM
Tris-HCl, pH 7.4, 25 mM NaCl. The buffer also contained 1 mM 2-mercaptoethanol (previously determined not to alter
the CD spectrum of TTK) in measurements of apo-TTK at neutral pH and in
titrations of TTK with cadmium or an excess of zinc (from stock
solutions of 10-100 mM CdCl2 or
ZnCl2).
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RESULTS |
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General Characteristics of Expressed TTK Peptide-- The TTK peptide was isolated from inclusion bodies and then renatured and purified in the zinc-bound form. SDS-polyacrylamide gel electrophoresis indicated a single band of the expected molecular weight. The reconstructed ESI-MS spectra obtained under acidic and neutral conditions indicated the presence of a single major species corresponding to the masses of the metal-free apo-TTK under acidic conditions (observed mass, 8023.5; expected mass, 8025.3) and TTK with two zinc ions (hereafter referred to as TTK) under neutral conditions (observed mass, 8151.7; expected mass, 8152.0). When dialyzed against ammonium acetate prior to ESI-MS, minor peaks indicative of ammonia adducts were also detected under both acidic and neutral conditions and presumed to be artifacts of reactions with ammonium acetate; these peaks were not detected if apo-TTK was prepared by acidification and desalting and analyzed directly without prior dialysis against ammonium acetate.
Inhibition of TTK-DNA Complex Formation by Thionein and Restoration by Zn-MT-- In titrations of 10 nM binding site DNA with different concentrations of the TTK peptide, band shifts were concentration-dependent with no detectable shift due to TTK-DNA complex formation in the absence of the peptide and a maximal shift at 12 µM peptide, a 1200-fold molar excess over binding site DNA. On subsequent gels, binding site DNA alone and binding site DNA plus 12 µM TTK peptide were used as negative and positive controls, respectively. The addition of unlabeled binding site DNA as a competitor resulted in a concentration-dependent inhibition of the band shift (not shown) and confirmed the previously reported specificity of this peptide for the binding site DNA (24).
Incubation of TTK with thionein resulted in a concentration-dependent inhibition of TTK-DNA complex formation at concentrations of thionein ranging from 0 to 300 µM (Fig. 1). However, a relatively high concentration of 300 µM thionein, corresponding to a 25-fold molar excess of thionein to TTK, was required to achieve complete inhibition of the TTK-DNA complex formation. A concentration between 37.5 and 75 µM thionein, which was equivalent to 3.1- and 6.3-fold molar excess, was needed for 50% inhibition. These findings were in agreement with earlier reports (12, 13) that thionein is capable of inhibiting DNA binding by Sp1 and TFIIIA, zinc transcription factors with similar CCHH motifs.
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Inhibition of DNA Binding by Cd-TTK and Reversal by Zn-MT-- To assess the effect of cadmium substitution on DNA binding and whether incubation of Zn-MT with cadmium-reconstituted Cd-TTK has any effect, the apparent dissociation constant, K'd, for complex formation with DNA was estimated by analyzing a range of concentrations of TTK, Cd-TTK, and Cd-TTK + 1× Zn-MT in band shift gels. Four independent measurements were made for the K'd of TTK. Single lanes that showed responses at equivalent concentrations of 6 µM for TTK, Cd-TTK, and Cd-TTK + 1× Zn-MT were selected for presentation in Fig. 4. DNA binding by Cd-TTK (K'd = 5.3 µM) was significantly reduced (p < 0.0001) in comparison with the TTK peptide bound to two Zn2+ ions (K'd = 0.5 µM ± 0.1 (S.D.; n = 4). This estimate of K'd for TTK agreed with a previously reported value of 0.4 µM (24). The K'd for Cd-TTK, indicating a 10.6-fold weaker binding to binding site DNA, was considered conservative, due to the possibility that low levels of background Zn2+ in the band shifts may have contributed to the formation of a small amount of folded TTK as described for band shifts with apo-TTK. Incubation of Cd-TTK with equimolar Zn-MT reduced the K'd to 0.9 µM, which was not significantly different from that of TTK (p > 0.05). This restoration of DNA binding activity to the previously inhibited Cd-TTK was probably due to zinc-cadmium exchange and recovery of secondary structure to that expected for the zinc-bound TTK.
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Effect of Zn2+ Ions on the Circular Dichroic Spectra of
TTK--
The CD spectrum of the native TTK peptide at pH 7.4, with
negative maxima at 208 and 222 nm (Fig.
5, solid line), was
consistent with a peptide conformation with significant -helical
structure components. A structural composition of 34.5%
-helix,
36.5%
-sheet, and 29% random coil obtained by secondary structure
analysis (35) of this CD spectrum agreed with the 33%
-helix and
30%
-sheet structure of TTK obtained by x-ray crystallography of
the TTK-DNA complex (29). The CD spectrum of apo-TTK (Fig. 5,
dashed line), with a negative maximum at about
204 nm, showed features mainly of
-sheet and random coil structure,
with low
-helical content (below 10% in this case). The addition of
Zn2+ to apo-TTK at neutral pH significantly increased the
negative maximum at 222 nm, signifying an increased
-helical
structure (not shown). When TTK was prepared by adding Zn2+
to apo-TTK at pH 2 and neutralizing to pH 7.3, the CD spectrum of the
zinc-reconstituted TTK (Fig. 5, dotted line)
resembled that of the native TTK. Thus, the folding of the TTK zinc
finger domains to the native structure was coupled to the binding of zinc, a property also reported for other zinc finger domain peptides (36).
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Electronic Absorption and CD Spectroscopy of TTK Titrated with Cd2+-- The electronic absorption spectrum of the TTK peptide, with a maximum at 277 nm and a shoulder at 283 nm, is diagnostic of the high tyrosine content (4 Tyr) and the absence of tryptophan (Fig. 6). The binding of Cd2+ to Cys-thiolate ligands in peptides and proteins can be followed by monitoring the absorption intensity of the Cys-S-Cd(II) ligand-metal charge-transfer transitions between 240 and 250 nm (37, 38). Titration of TTK (Fig. 6A, curve a) with Cd2+ (Fig. 6A, curves b-e) resulted in incremental increases in the CysS-Cd(II) charge transfer transitions in the lower wavelength range of the absorption spectrum. The difference absorption spectra (Fig. 6A, inset), obtained by subtracting the TTK spectrum (curve a) from the cadmium-induced spectra b-e, accentuated increases in the absorption band associated with the Cys-S-Cd(II) charge transfer transitions. This titration experiment showed that Cd2+ is able to replace the Zn2+ ions from the Cys ligands of the zinc finger CCHH motif in TTK. However, the binding constant for the Cd2+ appears to be lower than expected from the relative affinities of Zn2+ and Cd2+ for simple inorganic N and S ligands, since a 150-fold molar excess of Cd2+ over Zn2+ was not sufficient to completely replace the zinc.
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Interaction of Cd-TTK with Zn-MT--
The effect of Zn-MT on the
structure of Cd-TTK is shown in Fig. 8.
When Zn-MT was added to Cd-TTK so that concentrations of zinc and
cadmium were equimolar, there was a shift in the CD spectrum from that
of Cd-TTK (dotted line) to one that showed
changes indicative of recovery of the -helical bands present in
zinc-bound TTK, i.e. decrease at 222 nm and increase at 193 nm (dashed line), after 35 min. To isolate the
TTK component of the composite spectrum derived from the TTK-Zn-MT
mixture, the spectrum of Zn-MT incubated with Cd2+
(thin solid line) was subtracted from
that of the mixture (dashed line). The resulting
difference CD spectrum (thick solid
line) closely resembled the CD spectrum of the native
zinc-bound TTK peptide of Fig. 5. These results demonstrated that
MT-bound zinc had exchanged for the cadmium bound to Cd-TTK, resulting
in full recovery of the secondary structure native to the zinc-bound
form of the peptide. This was consistent with the full recovery of DNA
binding activity after Cd-TTK was incubated with equimolar Zn-MT as
previously shown in Fig. 4.
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DISCUSSION |
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The kinetic lability of zinc binding sites (4, 5) confers on MT the potential for intermolecular interactions through metal exchange with other structures, despite the high thermodynamic stability of metal-MT complexes. Early studies (10) demonstrating that Zn-MT is capable of activating Zn-metalloenzymes provided the initial evidence for zinc transfer from MT to potential acceptor molecules. Of the various types of zinc-dependent proteins, those with relatively facile zinc exchange would be suitable candidates to enter into dynamic metal exchange interactions with MT (13, 17). Metalloproteins with zinc atoms bound in cluster or finger motifs, such as MT and certain zinc transcription factors, exhibit the most rapid zinc exchange rates (18, 39). Thus, the reports that thionein was capable of inhibiting sequence-specific DNA binding by the CCHH zinc finger proteins Sp1 and TFIIIA (12, 13) were of interest, since they implicated thionein in a regulatory function involving abstraction of zinc from such proteins. Our observation that removal of zinc abolished the ordered secondary structure of the TTK peptide agreed with results for other zinc finger structures in which zinc-dependent folding of the peptides had been established (36). The inhibitory effect of thionein on the TTK-DNA complex formation was therefore not unexpected and was consistent with these earlier reports. The reverse reaction, i.e. the activation of metal-depleted forms of CCHH zinc finger proteins by Zn-MT, had not been previously reported, however. Zn-MT, for example, was not able to activate apo-Sp1 in Xenopus laevis oocytes treated with EDTA (40). This would limit the role of MT to abstracting zinc and inhibiting DNA binding. However, our experiments clearly demonstrated that Zn-MT can activate apo-TTK and, thus, extended observations of Zn-MT-mediated activation of DNA binding to CCHH zinc finger proteins. This would take full advantage of the ability of the Zn-MT-thionein conjugate pair to act as a zinc donor-acceptor mechanism.
Thermodynamic considerations provide some insight into the energetic
feasibility of reciprocal Zn2+ transfers between Zn
transcription factors and MT, both with kinetically labile
Zn2+. For example, zinc exchange between CCCC zinc
transcription factors and Zn-MT is expected to be relatively facile
based on the similarity in zinc binding affinities reported to date:
e.g. KS = 9 × 1011
M1 for a model CCCC zinc finger peptide (15)
and 2 × 1012 M
1 for Zn-MT
(17). This is consistent with a report describing Zn-MT/thionein-mediated activation-deactivation of the estrogen receptor (14). For CCHH zinc finger peptides and proteins, the apparent
stability constants for Zn2+ range from lower values of
3.6 × 108 M
1 for the second
finger in TFIIIA (41), and 2 × 109
M
1 for the third finger of Sp1 (16) to higher
values of 2 × 1011 M
1 for a
designed consensus single finger peptide (Cp-1) (15) and 5 × 1011 M
1 for intact TF111A (17). These
considerations provide an explanation for lack of activation of Sp1 by
Zn-MT (40) based on the greater affinity of zinc for MT over Sp1. The
stability constant for Zn2+ in TTK is not known, although
our results suggested that the value for TTK should be greater than
that for Sp1 and similar or possibly higher than that for the synthetic
Cp-1 zinc finger (15). Zn-MT to apo-TTK ratios less than unity were
effective in completely restoring DNA binding, attesting to the
efficiency of this activation. However, because the levels of thionein
required to inhibit DNA binding by TTK were high (3-6-fold molar
excess of thionein over TTK for 50% inhibition), it appeared that the reciprocal reactions were not equivalent. TTK appeared to have an
unexpectedly high avidity for zinc in comparison with MT.
An alternative hypothesis (7-9) uncouples thermodynamic barriers from the zinc transfer process in proposing a mechanism for coupled zinc exchange between Zn-MT/thionein and apo/Zn-metalloenzymes with much lower Zn2+ affinities. It is based on observations that 1) the redox activity of MT thiols and their interaction with other redox couples such as GSH-GSSG can facilitate zinc release from Zn-MT by oxidation of metal-thiolate bonds and stabilization of the liberated thiol group by disulfide bond formation and that 2) zinc transfer from zinc metalloproteins to thionein can be facilitated by the presence of chelators and cellular agents such as Tris, citrate, and GSH. The presence of mercaptoethanol in our TTK samples may have mimicked some of the conditions attributed to GSH during incubations of TTK with thionein (0.5 mM mercaptoethanol during incubation), although effects of the reducing agent on zinc transfer were not apparent.
Both MT-bound Zn2+ and "free" Zn2+ were equally effective in activating apo-TTK at concentrations equivalent to 1 molar ratio of Zn2+ to the number of binding sites on apo-TTK. This observation argues against, but does not preclude, direct molecular interactions between TTK and MT as the basis for the metal exchange and is consistent with a role for MT in regulating the availability of free zinc (Reaction 1),
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Other metals can also bind zinc finger peptides. Substitution of cobalt
for zinc, for example, has been used to probe structural characteristics of the zinc finger motif (15, 36). Binding of
Cd2+ to CCHH zinc finger proteins has had varied results
with regard to effects on structural and functional properties. Cadmium
substitutes stoichiometrically for zinc in a three-finger peptide of
Sp1 (45), resulting in a form that exhibits both Cys-S-Cd(II) charge
transfer bands and full recognition of the cognate DNA (45), although the affinity for the DNA binding site appears reduced. Cobalt, nickel,
and manganese are also able to restore DNA binding to Sp1 apoprotein
(44), with nickel and manganese less effective than zinc, cobalt, and
cadmium. With a zinc finger peptide of human immunodeficiency virus-1
integrase, titration of the apopeptide with zinc, cobalt, and cadmium
results in similar CD spectra with a high degree of ordered structure
(32% -helical content) not seen in the metal-free peptide (46).
These findings are not in accord with our results with the two-finger
TTK peptide, which showed that stoichiometric substitution of cadmium
for zinc disrupted the ordered secondary structure normally displayed
by the zinc-bound form and inhibited its ability to bind DNA.
Substituting Cd2+ for Zn2+ by either titrating
TTK with Cd2+ or reconstituting Cd-TTK from apo-TTK
resulted in the appearance of characteristic Cys-S-Cd(II) charge
transfer bands and a large decrease in the -helical content. We
suspect that either one or both His ligands of the CCHH motif may not
have been involved in Cd2+ binding. This would result in an
uncharacteristic metal coordination environment and disruption of the
TTK-DNA contact points that reside in the
-helix of the zinc finger
motif (29). The reverse titration, i.e. titrating Cd-TTK
with Zn2+, indicated a preference for Zn2+ over
Cd2+ that was consistent with the rank order of stability
constants for the consensus single finger peptide Cp-1 of 2 × 1011 M
1 for zinc and 5 × 108 M
1 for cadmium (calculated
from Ref. 15).
Since incubation of TFIIIA with cadmium also inhibited sequence-specific DNA recognition (23), it appears that TTK and TFIIIA have characteristics in common with regard to response to cadmium, which differ from those of Sp1, although the zinc fingers of all three are characterized by the CCHH motif. This may reflect an influence of other aspects of secondary structure on the metal coordination environment of zinc finger motifs of different proteins. Zinc binding to a three-zinc finger peptide of Sp1 is reported to be cooperative (45), while the zinc affinity of a synthetic zinc finger was increased when arranged in tandem with a like motif (47). Given the large numbers of CCHH zinc finger proteins (48) and the specificity attributed to their ability to bind DNA, diversity in their metal binding characteristics probably exists, possibly reflecting differences in zinc-mediated regulation of DNA binding.
Cadmium-induced alterations in the secondary structure of TTK and inhibition of DNA binding were reversed by incubation with stoichiometic amounts of Zn-MT. Measurements of CD indicated a complete and rapid exchange of Cd2+ and Zn2+ ions between Cd-TTK and Zn-MT. This metal-metal exchange is consistent with the much higher affinity of MT for Cd2+ over Zn2+ (49) and the preference of TTK for binding Zn2+ over Cd2+, indicated by titration of Cd-TTK with Zn2+.
These findings demonstrate the feasibility of an active role for Zn-MT in metal detoxification through metal-metal exchange reactions with adversely affected target structures. The unique properties of high thermodynamic stabilty and high kinetic lability of metal-binding sites, together with the order of affinity of different metals for MT (e.g. Hg(II) > Cd(II) > Zn(II)) (3, 18), confer on MT a unique ability to "rescue" or "repair" (21, 22) structures that have been compromised by inappropriately binding toxic metals with a greater affinity for MT than zinc. In this way, the Zn-MT-thionein conjugate pair, whose primary role may be to regulate zinc availability, may be diverted to serve detoxification functions when necessary.
Although MT is generally considered to be cytosolic, its localization in the nucleus is known for several cell types and species (50-52). Concentration of MT in the nucleus is reported to be energy-dependent and a means for supplying zinc to this organelle (53). These studies place MT at a site with a high requirement for zinc and also where pathological conditions associated with cadmium-induced genotoxicity or carcinogenesis (54) and interactions with electrophiles such as antitumor drugs (55) can occur. Furthermore, thionein is reported to exist in cells in at least one study (56). Our study described mechanisms whereby Zn-MT and thionein may interact with structures within the nucleus for the purpose of zinc regulation and metal detoxification.
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ACKNOWLEDGEMENTS |
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Special thanks are extended to Dr. L. Fairall
(Medical Research Council, UK) for the gift of the 911zf clone and
advice on its expression; Dr. G. Caderas and Prof. Dr. B. Gutte
(University of Zürich) for providing facilities and advice for
conducting band shift gels; Peter Faller (University of Zürich)
for assisting in MT preparations; and Dr. Peter Gehrig (University of
Zürich) for conducting the ESI-MS analyses.
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FOOTNOTES |
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* This study was supported in part by Fogarty International Center, National Institutes of Health Grant 1 F06 TW02150-1A1, Swiss National Science Foundation Grant 31-49460.96, and Canton Zürich.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, P.O. Box 38, Solomons, MD 20688. Tel.: 410-326-7235; Fax: 410-326-7210; E-mail: groes{at}cbl.umces.edu.
1 The abbreviations used are: MT, metallothionein; MES, 2-(N-morpholino)ethanesulfonic acid; DTT, dithiothreitol; K'd, apparent dissociation constant; ESI-MS, electrospray ionization-mass spectrometry; TTK, Tramtrack; CCHH, 2 Cys, 2 His; CCCC, 4 Cys.
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REFERENCES |
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