Chem. Res. Toxicol. 1995,8, 1020-1028
1020
Properties of the Spl Zinc Finger 3 Peptide: Coordination Chemistry, Redox Reactions, and Metal Binding Competition with Metallothionein Matthew C. Posewitz and Dean E. Wilcox* Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755-3564 Received March 13, 1995@
Toxic and/or carcinogenic consequences may result from metal ion substitution for the Zn(11)in transcription factors containing zinc fingers, and the small Cys-rich metal-binding protein metallothionein (MT) may play a role in this metal substitution. To begin to evaluate this hypothesis, with regard to the carcinogenic metal ion Ni(II), a peptide corresponding to the third finger of the transcription factor S p l (Spl-3) has been synthesized and its metal binding and redox reactions have been studied. The peptide binds Zn(II), Co(II), and Ni(II), with spectroscopic data indicating a tetrahedral coordination for the latter two; metal ion affinities have been quantified (& = 6 (k3) x 10-lo, 3 (&1)x and 4 ( k l ) x respectively) and found to be less t h a n those of a n optimized zinc finger peptide (Krizek, B. A., Merkle, D. L., and Berg, J. M. (1993) Inorg. Chem. 32,937-940) but greater t h a n those of the second finger of transcription factor IIIA (Berg, J. M., and Merkle, D. L. (1989) J . A m . Chem. SOC.111, 3759-3761). Reactions of the peptide and its metal-bound forms with dioxygen or hydrogen peroxide did not produce oxygen radical species; however, oxidation of the two Spl-3 cysteines was modulated by metal ions (Zn < Co = apo < Ni), suggesting a protective role for Zn(I1) but a n enhancing role for Ni(I1). Metal binding competition between S p l - 3 and the a domain of human liver MT-2 (a-hMT2) indicates a similar affinity for Zn(I1). However, a-hMT2 has a higher affinity for Ni(II), suggesting t h a t MT may play a protective role by ensuring Zn(II), rather t h a n Ni(II), coordination to zinc finger sequences of transcription factors. substitution in zinc fingers (27). Yet, the chemical and biological consequences of metal substitution for zinc in Although discovered only 10 years ago ( 1 , 2 ) ,the zinc these transcription factors have received little attention. finger protein structure is now found to constitute the DNA-binding domain of numerous transcription factors A key role in cellular defense against metal ion toxicity is played by the small Cys-rich protein metallothionein ( 3 , 4 ) . Initially identified as tandem repeat sequences, (Tyr,Phe)-X-Cys-X2,4-Cys-X3-Phe-XS-Leu-X~-His-X~-~- (MT) (28-31), which consists of two metal-binding doHis (where X represents a variable amino acid), in mains, is induced by toxic levels of certain metal ions, transcription factor IIIA (TFII1A)l from Xenopus laeuis, and has a high affinity for a number of metals in the which requires zinc for binding to DNA (51, subsequent following order: Cu(1) > Hg(I1) > Cd(I1) > Zn(I1) > Costudies of individual zinc finger peptides demonstrated (11) > Ni(I1). Although recent experiments with transtetrahedral (Td) Zn(I1) coordination to the two conserved genic mice lacking the two major MT isoforms appears Cys and His residues and stabilization of a unique to rule out an essential role in development or reproducpeptide structure consisting of a ,L? hairpin and a helix tion (32, 33), other results suggest MT may play a role (6-10). More recent NMR and X-ray crystallographic in modulating cellular processes requiring zinc. MT has studies of these classical zinc fingers ( 1 1 -13) and other been shown to be capable of eliminating the ability of Zn(I1)-binding motifs found in transcription factors (14the transcription factors S p l ( 3 4 )and TFIIIA (35)to bind 16) have provided detailed insight about the specific to DNA, presumably by removing Zn(I1) and eliminating interactions that stabilize these protein-DNA complexes. the protein structure required for DNA recognition and Only a limited number of studies, however, have binding. focused on the interaction of these proteins and peptides Recently, we have begun to examine the role of with other metals ( 17-20). Besides Zn(II), certain metal metallothionein in the carcinogenesis of nickel (36-38) ions, most notably Co(I1) and Cd(II), bind to zinc finger and chromium (391, metal ions which cause oxidative peptides (19, 21 -24), and some confer DNA-binding damage to DNA. With regard to nickel, we began with ability to the transcription factor (25, 26). Sunderman the hypothesis that MT, induced by intracellular Ni(II), and Barber were the first to draw attention to the may remove Zn(I1) from transcription factors, thereby potentially toxic and carcinogenic effects of metal ion allowing Ni(I1) to bind to these proteins and resulting in altered DNA binding andor Ni(I1) redox reactions delAbstract published in Advance ACS Abstracts, August 15, 1995. eterious to DNA or the transcription factor itself. Abbreviations: TFIIIA = transcription factor IIIA of Xenopus laeuis, Td = tetrahedral, MT = metallothionein, Spl-3 = finger 3 In this study, we have begun to test key elements of peptide of human transcription factor Spl, a-hMT2 = a domain of this hypothesis through in vitro experiments. A peptide human liver metallothionein, Fmoc = (9-fluorenylmethoxylcarbonyl, corresponding to the third zinc finger of the transcription HMP = 4-(hydroxymethyl)phenoxy, TFA = trifluoroacetic acid, DTT = dithiothreitol, HEPES = N-(2-hydroxyethyl)piperazine-N'-2-ethanefactor S p l (Spl-3) (40) has been synthesized, its metal sulfonic acid, DMPO = 5,5-dimethylpyrroline l-oxide, CT = charge binding properties have been characterized and quantitransfer, CP-1 = consensus finger peptide (241, /3 = Bohr magneton fied, redox reactions of the metal-peptide complexes (BM), sh = shoulder.
Introduction
@
0893-228d95I2708-1020$09.00/0 0 1995 American Chemical Society
Chem. Res. Toxicol., Vol. 8, No. 8, 1995 1021
Spl Zinc Finger 3 Peptide
have been studied, and metal binding competition between this peptide and the a domain of human liver MT-2 (a-hMT2) has been examined.
Experimental Procedures Peptide Synthesis, Purification, and Characterization. The Spl-3 peptide, K-F-A-C-P-E-C-P-K-R-F-M-R-S-D-H-L-S-KH-I-K-T-H-Q-N-K,was synthesized with Fmoc-protected amino acids (41)on an HMP resin using an Applied Biosystems 431A automated peptide synthesizer. The final product was cleaved from the resin and deprotected with an aqueous 90% trifluoroacetic acid (TFA)and 5% phenol cleavage solution. The a-hMT2 peptide, K-S-C-C-S-C-C-P-V-G-C-A-K-C-A-Q-G-C-I-C-K-G-A-SZn-Spl-3 D-K-C-S-C-C-A,was synthesized as reported previously (42). Peptide cleavage and deprotection were performed by Chiron Mimotopes Peptide Systems (San Diego, CAI. The crude pepNi-Spl-3 tides were dissolved in 0.1 M Tris buffer (pH 8.0) solution containing 100 mM dithiothreitol (DTT) and purified by reverse phase HPLC with a C-8 preparative column (2.14 x 25 cm) and an aqueous acetonitrile solvent gradient containing 0.1% TFA. The peptides were purified to '95% and >97% purity, respectively, based on reverse phase HPLC peak area with a C-18 -12 I 1 I I i analytical column (0.46 x 25 cm), and shown to have the 220 240 260 280 300 320 expected mass spectrum (m/z = 3270.5; calcd H = 3269.4), Wavelength (nm) performed by Chiron Mimotopes Peptide Systems. Additional peptide purification to remove metal ion impurities Figure 1. CD spectra of 43 pM Spl-3 peptide and the peptide consisted of dissolving the peptide in 1.0 mM HC1, concentrating after addition of 65 pM Zn(II), Ni(II), or Co(I1). and rediluting the sample with 1.0 mM HC1 three times in an Amicon miniconcentrator equipped with a YM1 membrane (1000 Spin trapping experiments used EPR spectroscopy to detect amu cutom, and then relyophilizing the peptide. Metal binding the generation of oxygen radicals in reactions of the Spl-3 studies were performed in 50 mM HEPES buffer (pH 7.0) peptide and were performed with solutions consisting of 92 mM solution containing 50 mM NaCl. Buffer solutions were pre5,5-dimethylpyrroline 1-oxide (DMPO) (purified with activated pared fresh, treated with Chelex 100, thoroughly deoxygenated, carbon), 0.234 mM Spl-3, 0.250 mM Zn(I1) or Ni(II), 20 mM and purged with argon prior to use. Peptide concentrations HEPES, 50 mM NaCl, and 12 mM hydrogen peroxide. EPR were determined by quantifying free thiols with the reagent 2,2'measurements, consisting of the sum of 10 scans, were obtained dithiodipyridine, which binds to cysteine and releases the on a Bruker ESP-300 spectrometer operating at 100 kHz chromophore 2-thiopyridone (€343 = 7600 M-l cm-l) (43, 44). modulation frequency, 1.0 G modulation amplitude, 100 G sweep Metal Binding and Redox Reactions. Metal ion solutions width, 5.12 ms time constant, 21 s scan time, 2 mW microwave were prepared with NiS04, ZnSOd, and CoSO4. Ultravioletpower, and 9.771 GHz microwave frequency. After the addition visible absorption spectra were collected a t ambient temperature of hydrogen peroxide, an aliquot was transferred to a melting on a Perkin-Elmer 1-9 double-beam recording spectrophotompoint capillary inside a quartz EPR tube, and data were acquired eter. Circular dichroism spectra were obtained a t ambient 90 s after mixing and every 4 min thereafter for 30 min. temperature with an OLIS Cary 61 recording spectropolarimAdditional experiments were conducted with similar solutions eter. NMR spectra were obtained with a Varian Unity 300 MHz lacking hydrogen peroxide but presaturated with dioxygen. spectrometer equipped with a Sun Unix workstation. Samples Positive control experiments were conducted under identical for NMR spectroscopy were dissolved in 50 mM deuterated Tris conditions with Ni(I1) and the tripeptide Gly-Gly-His (49) and 50 mM deuterated sodium acetatem20 buffer (pD 6.8) replacing the Spl-3 peptide. solution containing 50 mM NaC1; the buffer solution was Experiments to quantify oxidation of the Spl-3 cysteines were thoroughly deoxygenated and treated with Chelex 100 prior to performed with 43 pM Spl-3 and 60 pM Zn(II), Co(II),or Ni(I1) use. Sample temperature was maintained at 21-23 "C, and in HEPES buffer (pH 7.0) solution containing 50 mM NaC1. solvent suppression was accomplished by decoupling the HDO After 4 h of exposure to ambient atmosphere, 300 pL of the resonance at 4.8 ppm, relative to TMS. peptide sample was added to 600 pL of 10 mM HCl and 100 pL The ambient temperature magnetic moment of Ni(I1) bound of an aqueous solution saturated with 2,2'-dithiodipyridine. The to the Spl-3 peptide was determined with the Evans method final solutions were found to have pH 3.5-3.6, which is low (45-47). The sample was dissolved in 100 mM deuterated Tris/ enough to dissociate metal ions from the peptide, and the 343 D2O buffer (pD 7.0-7.6) solution containing 50 mM NaC1, and nm absorption of 2-thiopyridonewas measured to determine the the HDO signal was used as the chemical shift marker. The number of reduced Cys residues (*4% error). molar magnetic susceptibility was calculated with the equation:
+
xhl=4jtfC3*f
x,r+
- 1]
where f i s the resonance frequency (Hz) of the NMR spectrometer, Afis the difference between the chemical shift (Hz) of the marker in the reference and in the sample solutions, c is the concentration of the sample in mol/mL, xo is the molar susceptibility of the reference solution, and d. and do are the densities of the sample and reference solutions, respectively (48). The effective magnetic moment, in units of Bohr magneton (p), was then calculated with the equation:
where T is the absolute temperature.
' ,
Results Initially, it was important to demonstrate that the synthetic Spl-3 peptide would bind metal ions. Figure 1 shows the UV CD spectrum of the apo (metal-free) peptide and the spectrum obtained upon addition of 1.5 equiv of ZnUI), Ni(II), or Co(I1). The overall negative ellipticity and lack of distinct features in the 210-240 nm region of the apopeptide spectrum indicates little if any secondary structure. Addition of Zn(I1) results in a distinct new band with negative ellipticity at 230 nm, consistent with CD spectra reported for other synthetic finger peptides upon addition of Zn(I1) (6, 21, 23), and indicates the development of secondary structure (i.e., a
1022 Chem. Res. Toxicol., Vol. 8, No. 8, 1995
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form of the peptide.
helix) associated with Zn(I1) binding to the peptide. Addition of Ni(I1) or Co(I1) also leads to new negative ellipticity in the 220-230 nm region and suggests a metal-dependent folding of the peptide upon binding these metal ions. In addition, the CD spectra of the Ni(11) and Co(I1) samples show additional features a t -305 (+) nm and a t -245 (+I, -280 (+I, and -300 (-1 nm, respectively; these are assigned as charge transfer (CT) transitions associated with the open shell metal ion.2 As shown in Figure 2, addition of Ni(I1) or Co(I1) to the Spl-3 peptide results in several new absorption bands in the near-UV and visible region. The intensity and energy of the bands a t 305, 380, and -400 (sh) nm for Ni(I1) and 290, -315 (sh), -340 (sh), and -370 (sh) nm for Co(I1) suggest they are thiolate-to-metal CT transitions associated with metal binding to a t least one of the two cysteines of the peptide. The visible transitions a t 565 and 625 nm of Co-Spl-3 are typical of d-d transitions of Co(I1) in a tetrahedral coordination, as found for Co(11) complexes with other classical Cysz-His2 finger peptides (6, 21, 23, 24). The visible spectrum of Ni-Spl-3 sample is less intense, but absorption bands are found a t 525 and 630 nm; they, in addition to the CT bands a t higher energy, are well resolved as positive and negative bands in the CD spectrum of the Ni(I1) sample (Figure 2, lower). In addition, the Ni(I1) peptide has a n absorption band a t 950 nm in the near-IR (data not shown). While not unambiguously indicative of Td Ni(I1) coordination, the 525 and 630 nm bands are similar in energy to the visible d-d bands reported for the Ni(I1) complex of the synthetic Cysz-His2 consensus finger peptide (CP1)prepared by Berg and co-workers (18). To quantify metal ion binding to the Spl-3 peptide, dissociation constants were determined by spectrophotometrically monitoring metal ion titrations (Figure 31, as outlined previously (17, 24). The Ni(I1) dissociation
* CT band(s)may contribute in the 220-230 nm region too, possibly contributing to the blue shift of the negative band attributed to a helix formation.
constant was determined by monitoring the intense CT transitions of the Ni(I1)-peptide complex (Figure 3A) upon addition of 5 pL aliquots of 5 mM Ni(I1) to 1mL of 122 pM peptide. The Co(I1) dissociation constant was determined by monitoring the strong d-d transitions of the Co(I1)-peptide complex (Figure 3B) upon addition of 10 p L aliquots of 1.1mM Co(I1) to 1 mL of 84 pM peptide. The Zn(I1)dissociation constant was determined by monitoring the loss of the strong d-d transitions of the Co(I1)-peptide complex (Figure 3C) upon addition of 10 p L aliquots of 1.1 mM Zn(I1) to 1 mL of 84 pM CoSpl-3. Kd values of 6 (h2) x 10-lo, 3 (+1)x and 4 ( f l )x were determined for Zn(II), Co(II), and Ni(111, respectively, using standard nonlinear least-squares methods (50)for fitting plots of the ratio of metal-bound peptide to total peptide versus metal added (Figure 3). Table 1compares these values to dissociation constants reported for other finger peptides. The 1-D 'H NMR spectrum of the Spl-3 peptide in the absence and presence of metal ions further characterizes metal binding to the synthetic peptide. The NMR spectrum of the apopeptide (Figure 4A) is similar to that reported for other synthetic finger peptides (6,8). Resonances from the two Phe and three His residues are expected in the aromatic region, and by comparison to the assignment of the NMR spectrum of the ADRla finger peptide (511, the broad multiplet a t 7.3 ppm is assigned as the resonances from the two Phe residues, and the 1:2 doublet a t 7.9 ppm and triplet a t 7.0 ppm are assigned as the Cc-H and Ch-H resonances of the histidines. In support of this assignment, integration of these features gives the expected 10:3:3 ratio, respectively. The NMR spectrum of this sample after addition of 1.1 equiv of Zn(I1) (Figure 4B) has distinct changes in the chemical shifts of resonances in the aromatic region. Primarily, this involves the appearance of features a t 6.20 and 6.44 ppm, but increased dispersion of resonances in both the aromatic and upfield regions also occurs upon addition of Zn(I1). This indicates a distinct folded structure is obtained upon binding Zn(I1) and is consistent with 1-D 'H NMR spectra obtained for other classical zinc finger peptides (6,8,24). We have not attempted a detailed assignment of these features or determination of the structure of the Zn-Spl-3 peptide in solution by 2-D NMR methods. Upon addition of Ni(I1) to the Spl-3 peptide under strictly anaerobic ~onditions,~ the NMR spectrum (Figure 4C) shows significant line broadening, suggesting a paramagnetic coordination for Ni(I1). In the aromatic region, the multiplet a t 7 . 3 ppm broadens, but the features at 7.9 and 7.0 ppm decrease in intensity and are lost as Ni(I1) is titrated into a peptide solution; the residual signals seen a t these chemical shifts in Figure 4C correlate with the 15%free peptide expected for 1mol equiv of Ni(II), based on the Ni(I1) Kd value (Table 1). Thus, Ni(I1) binding results not only in dipolar broadening of most peptide resonances, but specifically the loss of all three His resonances, either through contact shift or dipolar effect^.^ This contrasts with the IH NMR spectrum reported for the Ni(I1)complex of another finger peptide (18),where negligible broadening but numerous paramagnetically-shifted resonances are observed. Deuterated NMR buffer containing Ni(I1) was degassed on a vacuum line with five freeze-pump-thaw cycles, transferred to a glovebag filled with nitrogen, and added to an NMR tube containing the peptide. The NMR tube was then sealed, and the spectra were collected.
Chem. Res. Toxicol., Vol. 8, No. 8, 1995 1023
Spl Zinc Finger 3 Peptide A
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This study. Reference 17. References 18 and 19.
To quantify the magnetic properties of Ni(I1) bound to the peptide, Evans method measurements were made on the apopeptide and the Ni(I1)-peptide. To determine the diamagnetic contribution of the peptide and account for density corrections, data from the apo sample (8.29 mM Spl-3) were collected first, and the HDO peak was found to be shifted 1.7 Hz upfield. Addition of Ni(I1) to a final concentration of 5.12 mM, a stoichiometry where essentially all nickel is bound to the peptide, resulted in a 30.0 Hz downfield shift of the HDO peak. These measurements give a value of 3.4 PfNi for the room temperature magnetic moment of Ni(I1) bound to the Spl-3 peptide in the pH range 7.0-7.6. Redox reactions of zinc fingers and their complexes with transition metals may be physiologically important, and experiments were undertaken to characterize and quantify this chemistry. Initially, spin trapping experiments using DMPO and EPR spectroscopy (see Experimental Procedures) failed to detect the production of hydroxyl or superoxo radicals from reactions of hydrogen peroxide or dioxygen with the apo or the Zn(I1)- or Ni(11)-boundforms of the peptide. However, quantification of free Cys thiols after 4 h exposure to ambient atmosphere showed decreases of 6%, 16%, 18%, and 46% in the amount of reduced Cys in the Zn(II), apo, Co(II), and Ni(I1) forms of the Spl-3 peptide, respectively. These data indicate that Zn(I1) protects the peptide from oxidation but that Cys oxidation is enhanced by Ni(I1). To determine the metal ion affinity of this classical zinc finger peptide relative to that of metallothionein, metal Additional NMR data on the Ni-Spl-3 sample were acquired under conditions in which the HDO resonance was not decoupled. The aromatic resonances from the three His are again not observed, suggesting that saturation transfer from the solvent is not responsible for the loss of these resonances.
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Figure 4. ‘H NMR spectra of (A) 3.5 mM Spl-3 peptide; (B) this peptide sample after addition of 1.1equiv of Zn(I1); (C) 3.0 mM Spl-3 peptide after the addition of 1.0 equiv of Ni(I1).
competition experiments with the Spl-3 peptide and a-hMT2 were undertaken. Since a-hMT2 binds four dipositive metal ions, peptide concentrations were adjusted to provide equimolar metal binding sites (Le., [Spl31 = 4[a-hMT2]). Since MT lacks aromatic amino acids, only lH NMR resonances from Spl-3 are observed in the aromatic region of the NMR spectrum in these experiments. Figure 5B shows the lH NMR spectrum of a sample consisting of 400 pL of 0.842 mM Zn3.8-a-hMT2 and 400 pL of 3.42 mM Spl-3. By comparison to the IH
1024 Chem. Res. Toxicol., Vol. 8, No. 8, 1995
Posewitz and Wilcox A
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Discussion Transcription factors that contain zinc finger motifs constitute a major class of DNA-binding proteins. The
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Figure 5. IH NMR spectra in the aromatic region (6.0-8.4 ppm) of (A) 3.5 mM Spl-3 peptide; (B) a mixture consisting of 1.71 mM Spl-3 peptide and 0.42 mM Zn3.8-a-hMT2; and (C) sample from (B) after addition of Ni(I1) to a final concentration of 1.9 mM.
NMR spectrum of the apopeptide (Figure 5A), the partial emergence of the 6.20 and 6.44 ppm resonances, which are diagnostic of Zn(I1) binding to Spl-3, shows that approximately half of the Spl-3 peptide binds Zn(I1) originally bound to a-hMT2. Thus, under these experimental conditions, Spl-3 and a-hMT2 have similar affinities for Zn(I1). Figure 5C shows that upon addition of Ni(I1) to this sample the NMR resonances a t 6.20 and 6.44 ppm increase, giving an NMR spectrum identical to that of the Zn(I1)-bound form of the Spl-3 peptide. Since these samples have reached equilibrium (see below), this result indicates that a-hMT2 has a higher affinity for Ni(I1) than does Spl-3. Similar metal competition experiments with the Spl-3 peptide and a-hMT2 were undertaken using absorption spectroscopy to monitor the intense and characteristic thiolate-to-Ni(I1) CT transitions associated with Ni(I1) binding to both peptides. The data in Figure 6A show that when 500 p L of 34 pM Zn4-a-hMT2 is mixed with 500 p L of 149 pM Ni-Spl-3 , the molar extinction coefficient increases and the spectrum changes immediately after mixing. The spectrum continues to increase for -45 min, resulting in an absorption spectrum similar in shape and intensity to that of Ni4-a-hMT2.As shown in Figure 6B, when the converse reaction is performed and 500 p L of 34 pM Ni4-a-hMT2 is mixed with 500 p L of 149 pM Zn-Spl-3, negligible change is observed in the absorption spectrum, which is predominately that of Ni4-a-hMT2. Thus, in agreement with the NMR data of Figure 5 , these results also indicate that a-hMT2 has a higher affinity for Ni(I1) than does the Spl-3 peptide.
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Zn-Spl-3 at t=45 minutes
-
-z
4000-
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250
300
350
400
450
500
550
600
Wavelength (nm)
Figure 6. Absorption spectra of (A) 149 pM Ni-Spl-3 peptide, a mixture of 75 pM Ni-Spl-3 peptide and 17 pM Zn4-a-hMT2 1 min after mixing, and this sample 45 min later; and (B) 34 pM Nid-a-hMT2, and a mixture of 17 pM Ni4-a-hMT2 and 75 ,uM Zn-Spl-3 peptide 45 min after mixing.
DNA-binding domains of these proteins have conserved Cys and His residues in tandem repeat sequences that bind Zn(I1) and thereby stabilize a specific peptide structure with favorable interaction between specific protein amino acid residues and DNA bases of the cognate binding site. However, if a different metal ion replaces Zn(1I) in these proteins, the zinc finger peptide structure may be altered, DNA binding may be compromised, and deleterious consequences for cell growth or survival may ensue. Another consequence of substituting a transition metal ion for Zn(I1) is metal-centered or metal-catalyzed redox reactions that may damage DNA or the transcription factor itself; these reactions may be important in metal toxicity and/or carcinogenicity.
Chem. Res. Toxicol., Vol. 8,No. 8,1995 1025
Spl Zinc Finger 3 Peptide Nickel is one of the few clearly documented carcinogenic metals (52). While Ni(I1) can cause oxidative damage to DNA (53),the molecular mechanism by which Ni(I1) induces cancer is not known. Therefore, it is possible that DNA damage relevant to nickel carcinogenesis may result from Ni(I1) substitution for the Zn(11)in transcription factor proteins. Although Ni(I1) has been reported to be ineffective in restoring the ability of TFIIIA (5))human TFIIIC (251,and estrogen receptor (54)to bind to their cognate DNA-binding site, the case of S p l is not so clear. Initial data suggested that neither Ni(I1) nor Co(I1) was able to restore sequence specific binding to DNA (401,but a later study showed Co(II), Cd(II), and, to a lesser extent, Ni(I1) capable of sustaining S p l binding to a n oligonucleotide containing the GC box sequence, its cognate binding site (26).Recent work with a peptide fragment containing the DNA-binding domain of S p l indicates that Ni(I1) is capable of providing DNA binding ability, but with some alteration of sequence specificity relative to that of Zn(I1) (55). Recent in vitro experiments with S p l (34)and TFIIIA (35)have suggested the possibility that metallothionein may be capable of removing zinc from transcription factors, thereby modulating their DNA-binding ability and the initiation of transcription. Thus, MT may play subtle but important roles in zinc homeostasis, and since Zn(I1) is required for a whole class of transcription factors, this could have major consequences for normal and abnormal cellular function. In light of these results, the interaction of MT with essential and with toxic or carcinogenic metals needs to be examined further, and we are investigating the case of Ni(I1). Certain welldocumented observations led to a working and testable hypothesis for the role of metallothionein in Ni carcinogenesis: (1)carcinogenic nickel solids are phagocytized by cells and release Ni(II), leading to increased intracellular nickel concentrations (37,56);(2) elevated levels of nickel are found in the nucleus under these conditions (37);(3) in certain cell types Ni(I1) induces the biosynthesis of MT (57);and (4) MT has a higher affinity for Zn(I1) than Ni(I1) (58). On the basis of these observations, we set out to (1j test whether MT is able to remove Zn(I1)from zinc finger sequences and allow Ni(I1) to bind to transcription factor proteins, and (2) study the structure and reactivity of Ni(I1) bound to zinc finger sequences. Toward these goals, we synthesized a peptide corresponding to the third finger of the transcription factor S p l and studied its properties. This classical zinc finger is believed to bind Zn(I1) with two Cys and two His in a tetrahedral coordination. Initially, we used CD and lH NMR spectroscopy to characterize the properties of the apo and the Zn(I1)-bound form of the peptide. Addition of Zn(I1) causes a n expected change in the U V CD spectrum (Figure 1) and characteristic shifts in the aromatic region of the lH NMR spectrum (Figure 41, both indicative of new secondary structure upon binding Zn(II), as found for several synthetic zinc finger peptides (6,21, 23). Further, the affinity of the peptide for Zn(I1) has been determined to be somewhat greater than that of the second finger of TFIIIA (27)but lower than that of the CP-1 finger peptide (18,19).Thus, it appears that the synthetic finger 3 of S p l binds Zn(II), analogous to several other classical zinc fingers. Nickel Finger. Nickel(I1) binding to the Spl-3 peptide was studied with several physical methods and comparison to Zn(I1) and Co(I1) binding to the peptide. The latter was included because Co(I1) has a thermody-
namic propensity for tetrahedral coordination, has strong and characteristic d-d transitions indicative of Td coordination, and has been included in numerous studies of the metal binding properties of zinc fingers (6, 17,21, 23,24).Addition of Co(I1) to the Spl-3 peptide results in new intense bands in the near-UV, assigned as thiolate-to-Co(I1) CT transitions and indicative of Co(I1) binding to one or both of the Cys thiolates, and new strong bands in the visible region indicative of Td Co(I1) coordination (Figure 2). Co(I1)also causes changes in the UV CD spectrum of the peptide; these include new negative ellipticity in the 215-235 nm region, which includes the 230 nm band seen with Zn(II), in addition to other positive and negative bands that are most likely CT transitions of the open shell Co(I1) ion (Figure 1). We expected that Ni(I1) would bind to the peptide with a Cysz-His2 coordination similar to that of Zn(I1) and Co(11). Four-coordinate N U ) , however, is found in geometries ranging from Td to square planar, with a thermodynamic preference for the latter, depending on the ligands. These two limiting cases can be distinguished experimentally, since d8 electronic configurations are diamagnetic in a square planar coordination but paramagnetic in a tetrahedral geometry. Further, the actual magnitude of the magnetic moment gives some indication of the degree of distortion from Td symmetry or of a solution equilibrium with a square planar geometry; both result in a decrease in the magnetic moment from 4.2 P/Ni for rigorous Td symmetry. The room temperature magnetic moment of 3.4 PINi for Ni-Spl-3 is consistent with a somewhat distorted Td coordination for the Ni(I1) ion, as found for the structurally characterized -Td Ni(I1) complexes: bis(N-isopropyl-3-ethylsalicylaldiminato)Ni(II) (3.3 /?) (59,60))bis(imidotetramethyldithi0diphosphino-S,S)Ni(II) (3.4 P) (61, 621, and tetraphenylphosphine tetrakis(thiophenolato)Ni(II) (3.2 P) (63, 64). While the magnetic data cannot rule out an equilibrium between -Td and square planar coordination for Ni(I1) bound to the peptide in solution, lower magnetic moments typically are associated with this type of behavior (60,62,64). Experimental measurements of the temperature dependence of the Ni(I1) magnetic properties would help distinguish these two cases (621,although the temperature range available with aqueous protein solutions is limited. The bands observed a t 525, 630, and 950 nm in the absorption spectrum of Ni-Spl-3 constitute a t least part of the Ni(I1) ligand field spectrum and can be used to help determine the Ni(I1)coordination. In Td coordination, Ni(11) is expected to have three spin-allowed transitions: 3T1(P) 3T1(F),3Az 3T1(F),and 3Tz 3T1(F),typically found in the visible, near-IR, and far-IR, respectively. As the symmetry is distorted from Td, the orbital degeneracy of the 3T1ground state is removed, as is the degeneracy of the 3Tzand 3T1excited states. The -3200 cm-l energy difference between the 525 and 630 nm bands, thus, may be the splitting of the highest energy d-d transition and reflect the mixed ligand set and the distortion of the Ni(11) geometry from Td. A similar assignment has been made for the 670 and 720 nm bands of Ni-substituted rubredoxin (65))where the weaker ligand field of the four cysteines shifts the bands to lower energy and leads to a smaller splitting (-1000 cm-l), relative to the Cysz-His2 ligation expected for Ni-Spl-3. The 950 nm band may be a component of the 3Az 3T1(F) transition, but additional absorption bands to lower energy cannot be resolved reliably from water overtone bands,5 even in DzO solvent, At least for the 525 and 630 nm bands, however,
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1026 Chem. Res. Toxicol., Vol. 8, No. 8, 1995
there is good agreement between the energies of the d-d transitions of Ni-Spl-3 and those of Ni-CP-1(18),indicating similar Ni(I1) coordination to the two finger peptides. This similarity is also seen for the energies of the CT transitions in the n e a r - W absorption spectrum. We have not been able to obtain as sharp and wellresolved 'H NMR data for the Ni(I1) complex of Spl-3 as that reported for the Ni(I1) complex of CP-1 (18) or for the -Td Ni(I1) substituted for the Zn(I1) in CdZn superoxide dismutase (66) or for the Fe(I1) in rubredoxin (67). This may be due to a lability of either the Ni(I1) coordination or the peptide conformation, which is not found in these other Ni(I1)-proteins. Future experiments aim to obtain improved spectral data by determining the effects of pH, peptide concentration, metal concentration, temperature, and ionic strength on the lH NMR spectrum of Ni-Spl-3. Nevertheless, Figure 4C and additional observations do provide some new insight about Ni(I1) coordination to the peptide. Addition of Ni(I1) results in loss of the lH resonances of all three His, including the one located adjacent to the conserved Leu and four residues toward the N-terminus from the first putative metal-binding His. This indicates a strong contact (through bond) or dipolar (through space) interaction with the paramagnetic metal. While this behavior is expected for the two histidines believed to be ligands for the Ni(II), it requires that the third His, located one turn away and on the same face of the a helix as the other two, be in close proximity to the Ni(I1) or possibly a directly-coordinated ligand. Although the latter explanation may seem unlikely, in light of magnetic and spectroscopic data indicating four-coordinate -Td Ni(I1) bound to the peptide, the data do not distinguish between a Cysz-His2 or a Cysl-His3 ligand set.6 Since a His in this same position in the middle finger of Zif268 has a specific hydrogen bond contact with a guanine in the middle of the recognition sequence (11) and a His-Tyr mutation of the His in this same position of the ADRlb finger eliminates ADR binding to DNA (8), Ni(I1) binding to this His in S p l finger 3 (or finger 1) would be expected to have a t least some effect on DNA recognition and binding by the transcription f a ~ t o r . ~ Although a recent study has shown that a peptide corresponding to the DNA-binding domain of S p l can be reconstituted with Ni(I1) and does bind to the GC box, it does not bind as tightly and has altered specificity relative to that of the zinc peptide (55). The affinity of the Spl-3 peptide for Ni(I1) has been quantified and, as found for Zn(I1) and Co(II), is less than the Ni(I1) affinity of the optimized CP-1 peptide (Table 1). In addition, the affinity of Spl-3 for Zn(I1) and Co(11) is greater than that of finger 2 of TFIIIA. Makowski and Sunderman have measured the Zn(I1) and Ni(I1) affinity of the entire TFIIIA protein by equilibrium dialysis and report high and low affinity Kd values for each metal ion, which are 1.0 x lo-@and 2.6 x for Zn(II), and 2.3 x and 5.2 x for Ni(I1) (68). Even the higher of these average Zn(I1) affinities of the nine fingers of TFIIIA is less than that of the individual finger E Weak near-IR bands at 2. >1200 nm reported for Ni-CP-l(I8)and Ni-substituted rubredoxin (671, even in deuterated buffer solution, are suspect. The current spectral and magnetic data also cannot eliminate 5-coordinate Ni(II1 with a Cysz-Hiss ligand set. Future experimental work with a modified Spl-3 peptide will address the possibility of this coordination. CP-1, fingers 1 and 3 of Zif 268, and ADRla do not have a His at this position, but do have a potentially metal-binding carboxylate (Glu or Asp). ~
Posewitz and Wilcox 2 of this transcription factor (Table l),as determined by spectral titration (17). This suggests that either there can be quite a range of metal ion affinities among the zinc fingers of a transcription factor or there can be significant cooperativity in metal binding among the tandem repeating finger sequences (68). Spl-3 has a higher affinity for Ni(I1)than the average values reported for TFIIIA, consistent with the generally higher affinity of Spl-3 for metal ions, relative to that of finger 2 of TFIIIA (Table 1). Redox Reactions. Although Ni(I1) is found in a variety of 4-,5-, and 6-coordinate structures, the redox chemistry of this metal ion in aqueous solution is not extensive. The Ni(II1) Ni(I1) reduction potential is lowered by strong square planar ligand fields, making Ni(II1) accessible for certain complexes (69), most notably the tripeptide Gly-Gly-His, where Ni(I1) is bound by the N-terminal amine, two deprotonated amides, and the His imidazole (70). Nickel(I1) coordinated to this tripeptide participates in redox reactions with HzOz to generate oxygen radical species (49). Further, there have been recent reports of redox reactions of peroxides with Ni(I1) bound to Cys (71) and to the tripeptide glutathione (72). Extensive experimental work in this study found no evidence for the generation of oxygen radicals in reactions of Ni-Spl-3 with HzOz or dioxygen. However, oxidation of the peptide cysteines was found, and the degree of this protein oxidation depends upon the metal ion involved. This is an important result in light of recent work that showed that oxidation of Cys in the glucocorticoid receptor prevented DNA binding of this transcription factor, even in the presence of required Zn(I1) (73). It is perhaps somewhat reassuring, therefore, that Zn(I1) retards the oxidation of the Spl-3 peptide, relative to the apopeptide, suggesting it plays a protective role toward oxidation of the transcription factor. On the other hand, Ni(I1) enhances peptide oxidation, suggesting that this may be a deleterious role for Ni(I1) when it is bound to the zinc fingers of transcription factors. While we suspect that the loss of free thiols results from formation of intramolecular disulfide bonds in these experiments, we have not characterized the product(s1 of Cys oxidation nor sought evidence for the oxidation of other residues (i.e., histidine oxidation to 2-oxohistidine (74)). Metal Binding Competition. An important component of our working hypothesis of a role for MT in the carcinogenesis of Ni(I1) is the ability of MT to remove Zn(I1) from transcription factors and allow Ni(I1) to bind to zinc finger sequences. Since we have quantified the Zn(I1) and Ni(I1) affinity of Spl-3, this could be determined readily if metal ion affinities of MT were known. Unfortunately, MT affinities have been reported for only a few metals because of inherent difficulties in measuring the binding of multiple metal ions to a multi-thiolate peptide ligand (75). Thus, we needed to determine experimentally the relative Zn(I1) and Ni(I1) affinities of MT and Spl-3 to help evaluate this hypothesis. The a domain of MT was used in these in vitro experiments and should provide an upper limit for Zn(I1) affinity, since it binds Zn(I1) more tightly than the fl domain (76).@While numerous studies have examined the competition between metal ions for MT, studies involving the competition of MT and another protein for a metal ion are rare. Besides the recent and relevant studies of Zeng et al. (34,
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Recent metal binding competition experiments, analogous to those shown in Figure 6, involving whole rabbit liver MT provide similar results as obtained with a-hMT2.
S p l Zinc Finger 3 Peptide 35), competition reactions with certain zinc-containing enzymes indicate that Zn7MT can donate Zn(I1) to the apoenzyme if it has a higher amnity for Zn(I1) (77, 78). Our competitive metal binding results for a-hMT2 and Spl-3 at a 1:l ratio of metal binding sites indicate that these peptides have a similar affinity for Zn(I1). This can be compared with the results of a previous study of TFIIIA, where a 13-fold excess of apo MT was required to remove zinc and eliminate DNA binding by the transcription factor. However, the observation that additional Zn(I1) restored DNA binding by TFIIIA prior to MT becoming fully metal loaded, suggested that TFIIIA and MT appear to compete equally for Zn(I1) (35). Nevertheless, comparisons with this study should be made cautiously because of the different nature of the experiment and the different proteins involved. Since we have shown that a-hMT2 and Spl-3 have similar affinities for Zn(II),this infers an average & value of 6 x for a-hMT2. This direct measurement indicates a lower Zn(I1) affinity than that reported from indirect measurements on whole MT (average & = 5 x (75) and warrants further investigation as to the nature of this discrepancy. The in vitro competition experiments involving a-hMT2, Spl-3,Zn(II), and Ni(I1) have given the important result that MT has a higher affinity for Ni(I1) than does Spl-3, resulting in Ni(II), instead of Zn(II), preferentially bound to MT. This is contrary to our working hypothesis and suggests that MT may suppress Ni(I1) binding to zinc finger sequences and prevent deleterious structural or redox consequences. This competition, however, will be influenced by the relative concentrations of the four species involved. Further, the metal ion affinity of zinc finger sequences may differ quite dramatically; for example, Kd values differing by 7 orders of magnitude have been reported for Zn(I1)binding to TFIIIA (68,791. Thus, additional studies involving other zinc-binding domains from transcription factors are underway currently to evaluate their metal binding competition with metallothionein.
Acknowledgment. We are most grateful to Karen Wetterhahn for insightful comments, critical reading of the manuscript, and encouragement. We also thank Tom Ciardelli for his assistance in the preparation of synthetic peptides and Jon Kull for the synthesis of a-hMT2. This research was supported by NIH Grant CA61349.
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