Interaction of Cd2+ with Zn Finger 3 of Transcription Factor IIIA

Finger 3 of transcription factor IIIA of Xenopus laevis was synthesized and constituted with. Zn2+ or Cd2+. The C-block element of the internal contro...
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Chem. Res. Toxicol. 2004, 17, 863-870

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Interaction of Cd2+ with Zn Finger 3 of Transcription Factor IIIA: Structures and Binding to Cognate DNA Dmitriy Krepkiy, F. Holger Fo¨rsterling, and David H. Petering* Department of Chemistry, University of WisconsinsMilwaukee, P.O. Box 413, Milwaukee, Wisconsin 53201 Received December 1, 2003

Finger 3 of transcription factor IIIA of Xenopus laevis was synthesized and constituted with Zn2+ or Cd2+. The C-block element of the internal control region of the promoter of the 5S rRNA gene binds to the Zn-F3 and Cd-F3 with dissociation constants of 2.6 × 10-5 and 1.5 × 10-4 M, respectively. According to NMR spectroscopy, Zn-F3, as well as Cd-F3, exists as a conformational equilibrium that is not susceptible to structural analysis by NMR methods. To restrict the observed conformational flexibility, a mutant F3 (mF3), which differs from F3 in the number and type of amino acids between the cysteine and the histidine ligands, was synthesized. The affinity of Zn-mF3 for the C-block DNA was greatly reduced relative to ZnF3. Nevertheless, the metal ion dissociation constants of the Zn- and Cd-mF3 complexes remain similar to those of the native structures at 4.5 × 10-9 and 3.2 × 10-8 M, respectively. Zn-mF3 is more thermally stable than Cd-mF3, but both adopt similar conformations according to twodimensional 1H NMR spectroscopy. Each peptide displays a ββR fold for its backbone that is typical of this class of zinc finger domains. The113Cd ion in 113Cd-mF3 is coupled to the protons of two cysteine and two histidine residues and characterized by a chemical shift of 567 ppm.

Introduction Zinc finger proteins comprise one of the most abundant classes of proteins in eukaryotic genomes (1). TFIIIA1 from Xenopus laevis is a prototypical structure, which contains nine zinc finger motifs of the two His, two Cys type (2, 3). Each zinc finger folds into a small domain with R-helix and antiparallel β-sheet structural features (4-8). The metal is coordinated by two histidine residues positioned within the R-helix and two cysteine residues located in the β-sheet. The folding of the domain is induced by the metal binding and includes the formation of a minihydrophobic core involving three side chain groups (9). The solution NMR structure of the first three fingers of TFIIIA bound to DNA was obtained in the laboratory of Wright (6, 10-12) and later confirmed by X-ray diffraction studies with a six finger fragment (8). Zn-TFIIIA binds to the ICR of the 5S rRNA gene and acts as a positive trans-acting element (3, 10, 13, 14). The ICR corresponds to the nucleotides +50 to +92 of the promoter sequence. On the basis of its interactions with Zn-TFIIIA, it is divided into three regions: block A (5064), an intermediate element (67-72), and the C-block (80-97), which interacts with fingers 1-3 (15). The C-block makes the greatest contribution to the binding affinity of the ICR for Zn-TFIIIA (16). Among fingers 1-3, F3 makes the largest number of contacts with DNA and, thereby, provides the most stabilization for the DNA adduct (6, 17, 18). * To whom correspondence should be addressed. Tel: 414-229-5853. E-mail: [email protected]. 1 Abbreviations: DQF-COSY, double quantum filtered correlation spectroscopy; DTNB, 5,5′-dithiobis(2-nitrobenzoate); EMSA, electrophoretic mobility shift assay; F3, finger 3; HSQC, heteronuclear single quantum correlation; HSED, heteronuclear spin-echo difference; ICR, internal control region; mF3, mutant finger 3; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TFIIIA, transcription factor IIIA; TOCSY, total correlation spectroscopy.

The zinc binding site in zinc finger proteins has been proposed as a plausible target for different toxic metals (19-23). It has been shown recently that the toxic metal ion, Cd2+, inhibits the specific interaction of Zn-TFIIIA with ICR DNA (24, 25). Similarly, Cd-tramtrak has at least a 10-fold lower affinity for its cognate DNA base sequence than Zn-tramtrak because it has lost much of the R-helical structure of the native protein (26). In contrast, the substitution of Cd2+ for Zn2+ in Sp1 does not alter qualitatively its DNA binding affinity (21, 27). In the current paper, the properties of Zn- and Cd-F3 of the TFIIIA were examined as a model to understand how Cd2+ reduces the affinity of TFIIIA for its cognate DNA binding sequence. Because of the fluxional nature of Znand Cd-F3, their structures could not be analyzed by NMR spectroscopy. Instead, structures of Zn2+ and Cd2+ bound to a mutant finger (mF3) with different numbers of interligand amino acids were determined in order to investigate the impact of Cd2+ substitution upon the three-dimensional (3D) conformation of a zinc finger.

Materials and Methods Synthesis of Native and Mutant Zinc F3 of TFIIIA from X. laevis. The peptide sequences of F3 of TFIIIA-KNFTCDSDGCDLRFTTKANMKKHFNRFHNI and mF3-KNFTCPECDLRFTTKANMKKHQRTHNI were made and purified by peptide synthesis as previously described (28). The underlined amino acids differ in the two sequences. To obtain the reduced apopeptide, 25% β-mercaptoethanol was incubated with the concentrated peptide solution under a N2 atmosphere for 48 h. The apo-peptide was purified by G-25 gel filtration chromatography (70 cm × 2 cm diameter column), using 0.01 N HCl. It was then concentrated with an Amicon concentrator (YM1 filter, 1000 MW cutoff) in an anaerobic atmosphere in the same medium. Tris buffer (10 mM) was added to the concentrated peptide, and the pH was adjusted anaerobically to 7.0. The concentration of the peptide’s sulfydryl groups was measured by reaction with

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DTNB (29). Using metal ion concentrations based on atomic absorption spectrophotometric measurements, a one-to-one ratio of Zn2+ or Cd2+ to apo-peptide was used to constitute the holoprotein. The integrity of the peptides was confirmed by SDS-PAGE amino acid analysis and MALDI-MS. The numbering of the amino acids and the sequence of the F3 peptide correspond to the generally accepted numbering of the amino acids of TFIIIA (8, 30). In numbering the mF3, number 1 corresponds to the first amino acid residue at the N terminus of the peptide. Determination of the Dissociation Constants for the Binding Reaction of Co2+, Cd2+, and Zn2+ with mF3. The affinity of Co(II) for apo-finger structures was determined spectrophotometrically by monitoring the appearance of the Co(II)-mF3 d-d bands between 550 and 700 nm during the anaerobic titration of apo-peptide with CoCl2 in 20 mM Tris/Cl and 250 mM NaCl, pH 7.4, at 25 °C as described elsewhere (4, 25). The apparent dissociation constants of the Zn- and Cdpeptides were determined by competitive titration of the Co(II)-peptide with ZnCl2 or CdCl2 in which the decrease in the absorbance spectrum of the Co-peptide was used to follow the formation of the Zn- or Cd-peptide (25). The apparent Kd at pH 7.4 was then calculated using the equilibrium:

Co(II)-peptide + Me2+ a Me-peptide + Co(II), Keq (1) where Keq ) ([Me-peptide][Co(II)])/([Co(II)-peptide][Me2+])

Keq ) KdCo(II)-peptide/KdMe-peptide

(2)

KdMe-peptide ) KdCo(II)-peptide/Keq

(3)

The concentrations of the metal ions were determined by atomic absorption spectroscopy. Calibration standards were prepared from commercial metal ion standards (1000 ppm) (Fisher Scientific). Synthesis and Purification of C-Block Promoter Element DNA Oligonucleotide. The DNA oligonucleotide, d(3′ACCTACCCTCTGG-5′,5′-TGGATGGGAGACC-3′), that comprises base pairs 80-92 of the ICR was synthesized using a Millipore Cyclone Plus DNA synthesizer. The synthesis was done on the standard 1.0 µmol scale using phosphoramidite DNA chemistry. The coding and noncoding strands were synthesized and then isolated, deprotected, and purified with HPLC and G-15 gel filtration chromatography, as previously described (31). Finally, the two strands were annealed to form the duplex. Electrophoretic Mobility Shift Experiments. The synthetic 13 base pair DNA oligomer was radiolabeled at the 5′ ends with 32P from ATP using T4 polynucleotide kinase (Promega) (32). For EMSA experiments, the 1:1 complex between the oligonucleotide and the Zn- or Cd-F3 or -mF3 was formed in a buffer comprised of 20 mM Tris/Cl, pH 7.4, 70 mM KCl, 10% glycerol, and 5 mM MgCl2 in the presence of 0.7 µg (40 µM in base pairs) of poly[d(I-C)] (Roche Diagnostics). The reactants were mixed by pipetting and incubated for 2 h at 37 °C. Before the mixtures were loaded into the well, 2 µL of loading dye was added. The peptide bound and unbound oligonucleotides were separated with 8% nondenaturing PAGE for 1.5 h in 89 mM Tris-borate buffer, pH 8.0, at 200 V. The gel was removed and dried and then placed in contact with X-ray film (Fuji) for 2 h at -70 °C. Afterward, the film was developed and the bands were quantitated with Kodak Digital Science 1 software. The dissociation constants for the Zn-F3 and Cd-F3 adducts with C-block DNA were calculated using the Grafit program (Erithacus Software Ltd.). NMR Spectroscopy. NMR data were acquired at 298 and 278 K on a Bruker DRX500 NMR spectrometer equipped with a broadband inverse triple axis gradient probe. The spectra of 1 mM peptide samples prepared in a N2 atmosphere were ordinarily recorded in 5 mM D11-Tris/Cl (CIL) and 100 mM NaCl buffer, pH 7.0, that contained 10% D2O. The proton chemical shifts were referenced to DSS using the water signal as a

Figure 1. Titration of the 13-mer C-block with Cd-F3. The conditions are as described in the Materials and Methods. Lanes 1-5: 4 µM 13-mer and Cd-F3 of different concentrations (300, 150, 75, 30, and 15 µM, respectively); lane 6, 25 µM Zn-F3. secondary internal reference. Two-dimensional (2D) DQF-COSY, TOCSY, and NOESY spectra were obtained by standard methods using flip back pulses and the WATERGATE scheme for water suppression (33, 34). In the NOESY spectra, mixing times of 100 and 200 ms were used; TOCSY spectra were acquired with a mixing time of 80 ms using a DIPSI-2 mixing scheme. States-TPPI-States phase cycling was used in all experiments for quadrature detection in the indirect dimension. Heteronuclear 1H{113Cd} HSQC experiments were performed with gradient selection employing a mixing time of 10 ms for the magnetization transfer and 32 complex points in the indirect dimension. The experiment was repeated with several offsets of the 113Cd frequency to ensure that no folding was observed. One-dimensional (1D) 1H{113Cd} HSED experiments were performed as described previously (35). Typically, 16 000 scans were accumulated with evolution times for the heteronuclear coupling ranging from 10 to 83 ms. The spectra were analyzed on a Silicon Graphics Octane workstation using Felix2000 software (Biosym Technologies Inc.). Zn-mF3 and Cd-mF3 NMR Assignments, Structural Constraints, and Structure Calculations. The assignment of most proton resonances was obtained using 2D TOCSY, COSY, and NOESY (100 and 200 ms) experiments. A total of 375 NOE restraints (145 intraresidual, 92 sequential, 78 medium-range, and 60 long-range) were obtained for Zn-mF3 and 276 for Cd-mF3 (Supporting Information). Measurement of 3JHNNR for Zn-mF3 yielded an additional eight dihedral angle restraints for the backbone angle φ. For the final refinement of the structures, 79 (Zn) and 74 (Cd) proton chemical shifts were included as restraints (36). The metal coordination was defined by adding four bonds, 12 angles, and six impropers involving the metal and the Cys and His residues using energy potentials of 300 kcal mol-1 Å-2, 100 kcal mol-1 rad-2, and 400 kcal mol-1 rad-2, respectively. Structure calculations were performed in the program ARIA1.2/CNS1.1 using the ab initio simulation annealing protocol and iterative calibration of distances and automated assignment of ambiguous NOEs (37, 38). One hundred structures were generated, and a subset of 20 of the lowest energy structures was selected for the final analysis. The final sets of structures exhibited no NOE violations > 0.5 Å.

Results Binding of Peptides to C-Block DNA. The quantitative binding of Cd- and Zn-F3 with the C-block oligonucleotide was investigated in EMSAs. In the absence of adjacent finger elements, ZnF3 forms a specific complex with the C-block promoter element (lane 6, Figure 1) (39). In comparison, the binding of Zn-mF3 to the C-block DNA was largely abolished. This effect is thought to be due to the lack of arginine in the interhistidine ligand region of the helix. This residue of F3 interacts specfically with the DNA according to the NMR structure of TFIIIA Zn fingers 1-3 bound to the C-block (6).

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Figure 2. 1H NMR spectra (500 MHz) of apo-F3 and F3 reconstituted with Cd2+ or Zn2+ in 5 mM D11-Tris/HCl and 100 mM NaCl, pH 7.0, with 10% D2O at 278 K. (a) Apo-F3, (b) CdF3, and (c) Zn-F3.

The affinity of Cd-F3 for the C-block oligonucleotide was measured (Figure 1). The 13-mer C-block oligonucleotide was titrated with 15-300 µM Cd-F3 (lanes 1-5). For comparison, 25 µM Zn-F3 was loaded in lane 6. Although Cd-F3 also displayed a significant capacity to bind to the C-block, it had a lower affinity for the oligonucleotide than Zn-F3 (300 µM Cd-F3 in lane 1 vs 25 µM Zn-F3 in lane 6). The interaction of Cd-F3 with the C-block DNA was characterized by a Kd of 1.5 ( 0.03 × 10-4 M (standard deviation of constant calculated from the set of points in the titration), whereas the Kd for ZnF3 binding to the C-block was 2.6 ( 0.1 × 10-5 M (data not shown). 1 H NMR Spectroscopy of Zn- and Cd-F3. To clarify the structure of the Zn-F3 peptide and explain the basis for the decrease in binding stability of the Cd-F3 at the C-block adduct, NMR experiments were conducted. The 1D 1H spectrum indicated that amide and aliphatic region peaks of apo-F3 were sharp and well-resolved (Figure 2). In addition, peaks were identified in the aromatic region, which corresponds to the two histidine δ2 and 1 protons and side chains of the four phenylalanines. Upon titration with Zn2+ or Cd2+, the spectrum was broadened but did not substantially change (Figure 2) (11). The 113Cd NMR resonance of 113Cd-F3 could not be detected. Furthermore, the cross-peaks of the NOESY and TOCSY spectra of the Zn- and Cd-F3 were broad and could not be assigned (data not shown). These results showed that each peptide was highly fluxional on the NMR time scale. Metal Ion Dissociation Constants of Zn-mF3 and Cd-mF3. The modest affinity of Zn2+ and Cd2+ for F3 was hypothesized to be responsible for the inability of Zn-F3 and Cd-F3 to form kinetically stable complexes. According to another study, the Kd(Zn-F3) ) 1.01 ( 0.03 × 10-8 M (average ( SD, three measurements) at pH 7.4 and 25° (25). The dissociation constant of Cd-F3 under the same conditions was also shown to be nearly equal to that of Zn-F3, 1.98 ( 0.04 × 10-8 M. In contrast, a consensus peptide, CP-1, that was defined over a decade ago is characterized by the zinc dissociation constant of 10-11 M (4, 40). It remains the sequence with the highest known affinity for zinc. The general sequence of the N2S2 zinc finger is (Tyr,Phe)-X-Cys-X2,4-Cys-X3-Phe-X5-Leu-X2-His-X3,4-His, where X represents variable amino acids (7). In an effort to

Figure 3. Amide region of 500 MHz 1 H NMR spectra of apomF3 and mF3 reconstituted with Cd2+ or Zn2+ in 5 mM D11Tris/HCl and 100 mM NaCl, pH 7.0, with 10% D2O. (a) ApomF3, (b) Zn-mF3, and (c) Cd-mF3. The H1 and Hδ2 of the His residues are highlighted. The highly shifted Phe12-Hζ is also indicated.

enhance the stability of F3 for Zn2+ and Cd2+, the numbers and types of amino acids interposed between the histidine and the cysteine residues in the native sequence were changed to those of CP-1 as shown in the Materials and Methods. The dissociation constants for Zn2+ and Cd2+ bound to mF3 were determined by backtitration of Co2+ reconstituted peptides with zinc or cadmium chloride, as described in the Materials and Methods. They were measured as 4.5 ( 0.15 × 10-9 M and 3.2 ( 0.1 × 10-8 M, respectively. Although small changes were detected in the Kd of Zn- and Cd-mF3 in comparison with the constants for the corresponding metallo-F3 complexes, they did not reveal a substantial increase in binding affinity, as had been hypothesized. Nevertheless, the NMR properties of both mutant metallo fingers were distinctly different from those of the Zn- and Cd-F3 complexes. NMR Spectroscopy of Zn-mF3 and Cd-mF3. ApomF3 readily folds and adopts stable conformations upon binding Zn2+ or Cd2+ (Figure 3). The NMR spectra of Znand Cd-mF3 were independent of the NaCl concentration between 0 and 250 mM. The spectra of Zn2+ reconstituted mF3 were practically independent of the temperature with only minor shifts in the spin system frequencies observed between 278 and 298 K. In contrast, the CdmF3 spectra changed dramatically over this temperature range. The peaks substantially broadened when the sample temperature was raised from 278 to 298 K (data not shown). For the structure calculations, spectra of ZnmF3 were recorded at 278 and 298 K; the spectra of CdmF3 were recorded at 278 K. The 1D NMR spectra of the unfolded apo-mF3 peptide include peaks in the aromatic region (Figure 3). The His21, H1 and Hδ2, and His25, H1 and Hδ2, protons were assigned to resonances at 7.60, 6.80 and 7.63, 6.90 ppm, respectively. Upon addition of Zn2+, these resonances correspondingly shifted to 7.78, 6.93 and 7.99, 6.66 ppm. Similarly, in the presence of Cd2+, His21, H1 and Hδ2, and His25, H1 and Hδ2, moved to 7.70, 6.90 and 7.86, 6.57 ppm, respectively. In addition, the signal of Phe-12:Hζ experiences a large upfield shift upon

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Figure 6. One-dimensional 1H{113Cd} HSED spectrum showing Cys-Hβ and His-H1/Hδ2 protons, which exhibit coupling to 113Cd in 5 mM D11-Tris/HCl and 100 mM NaCl, pH 7.0, with 10% D2O at 278 K. Mixing times were set for a coupling constant of 50 (A) and 10 Hz (B)

Figure 4. Summary of backbone NOE connectivities for (a) Zn- and (b) Cd-mF3. The width of the line corresponds to the intensity of the NOE observed (short-, medium-, and long-range NOEs).

Figure 5. Plot of the differences between the 1H chemical shifts of HR of random coil and Zn- and Cd-mF3 indicating secondary structure elements. Residues 15 and 18 exhibit additional shifts due to ring current effects.

addition of metal ion and is clearly separated in Zn-mF3 (6.27 ppm) and Cd-mF3 (6.12 ppm). The amino acid spin systems of the Zn- and Cd-mF3 were identified and assigned, using the 2D NOESY (100 and 200 ms mixing times), TOCSY, and COSY spectra (Table 1S). Met18 was identified by the specific upfield chemical shift of its R-proton, characteristic of zinc finger structures (12). Sequential assignments were obtained by using HN-HN and HN-HR regions of the NOESY spectra. Connections between adjacent residues were observed with a continuous path established between residues 3 and 27. The sequential NOE connectivities for Zn- and Cd-mF3 are shown in Figure 4. In these structures, the connectivities dRN(i,i+2), dRN(i,i+3), and dRN(i,i+4) indicate the formation of an R-helical structure in the region of residues 16-27 of the folded peptide (41). A bar graph of the differences between the 1H chemical shifts of HR of apo-F3 and of Zn or Cd-mF3 is portrayed in Figure 5. Negative shifts indicate an R-helical structure, and positive shifts are consistent with the β-sheet. The extreme 1H chemical shifts of conserved Lys15 and Met18

HR are consistent with other zinc finger NMR data and are caused by additional ring current effects (see below) (11, 42). The total number of NOEs defined for Cd-mF3 was smaller than for Zn-mF3 (Table 2S). However, for some residue pairs, NOEs are present only in the case of Cd-mF3 (Phe3-Phe12, Phe3-Thr14, Phe3-Lys15, Thr4Lys15, Glu7-Asn26, and Gln22-Ans26). It is thought that the smaller number of NOEs for Cd-mF3 in comparison with Zn-mF3 is that the Cd-peptide is more fluxional, leading to the observation of fewer NOE cross-peaks. The 2D 1H{113Cd} HSQC spectrum of 113Cd-mF3 was recorded. The chemical shift of the 113Cd was 567 ppm, consistent with the coordination of the Cd2+ to two nitrogens and two sulfur atoms (43). The temperature dependence of the chemical shift of 113Cd shows that the average chemical environment of the Cd2+ center changed somewhat with temperature. However, the perturbation was not dramatic (e5 ppm) and was within a range that implies the same coordination sphere over the temperature span. To observe the couplings of the 113Cd with protons of both the imidazole ligands of the histidines and the β-protons of the cysteines, 1D HSED experiments with different optimizations of the magnetization transfer were employed (35, 44). In Figure 6, the 1D 1H{113Cd} HSED spectra are shown exhibiting the Cys8-Hβ2, Cys5Hβ2, and C5Hβ2 of the cysteine residues and the His21H1, His21-Hδ2, His25-H1, and His25-Hδ2 of the histidines. It is clear that the coordination of the Cd to the mF3 occurs via the formation of bonds with two His and two Cys residues. Structures of Zn-mF3 and Cd-mF3. The structures of Zn-mF3 and Cd-mF3 were calculated using the ARIA/ CNS program as described in the Materials and Methods section. Twenty structure files were generated for each peptide. The overlaid structures of each metallo finger are displayed in Figure 7. An inspection shows that both of the individual structures for each peptide are strongly convergent, each describing the characteristic ββR conformation of C2H2 zinc fingers. Thus, the rmsd for the backbone of residues 3-26 of the ensemble of Zn-mF3 structures is 0.54 ( 0.16 Å and that for Cd-mF3 is 0.63 ( 0.15 Å. Furthermore, the rmsd for the backbone of the ensemble of Cd-mF3 structures in comparison with the mean Zn-mF3 structure is 2.15 ( 0.06 Å and that of the DNA binding helix (residues 12-25) is better at 1.67 (

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Figure 7. Stereview of the 20 lowest energy structures of Zn-mF3 (blue) and Cd-mF3 (red).

Figure 9. Relative position and orientation of Phe3 and Phe12 with respect to Lys15 and Met18 residues in Zn-mF3, indicating the sources for ring current shifts for Met18-HR and Lys15-HR. The number of side chain NOE restraints and side chain rmsd for the residues are Phe 3 (24, 1.34 Å), Phe 12 (36, 0.88 Å), Lys 15 (15, 0.91 Å), and Met 18 (25, 0.88 Å).

Figure 8. Backbone folds: structures of Zn-mF3 (A) and CdmF3 (B). The R-helical regions of the peptides are displayed as ribbons.

0.09 Å. Figure 8 shows the average ribbon structures of peptide, calculated by the MOLMOL program. Differences in the two structures can be seen in the parts of the helical region that attain full R-helical conformation. Although the qualitative folding of the two molecules is quite similar, small differences between them can be detected. Whether these perturbations are sufficient to explain why the substitution of Cd2+ for Zn2+ might adversely affect the capacity to make specific fingerDNA interactions is unresolved. Consistent with other zinc finger peptides, the R-protons of Lys15 and Met18 in Zn- and Cd-mF3 are shifted considerably (1-2 ppm) upfield relative to their random coil chemical shifts, more than expected by a shift induced by helical structure alone (Figure 4) (36). On the basis of the solution structure of the TFIIIA Zn finger 1-3, Liao et al. speculated that this behavior arises from the ring current effect of residues Phe94 and Phe97, which are positioned between histidines in the native F3

(10). Although these residues are not part of the mutant sequence, the significant upfield shift of the R-protons of Lys15 and Met18 is still observed. Analysis of NOE contacts and calculation of ring current shifts within the program MOLMOL using the method of Case et al. indicates that the upfield shifts instead arise from ring current shifts induced by the residues Phe3 and Phe12, respectively (Figure 9) (45, 46). Phe3 and Phe12 are conserved residues among the zinc finger peptides, which would explain the conserved shifts of the residues Lys15 and Met18 among all of the peptides studied (42). Calculations with both the solution and the crystal structures of TFIIIA/DNA complexes indicated the upfield shifts of Lys87 and Met90 to be caused by the conserved Phe73 and Phe84, respectively, which are equivalent to Phe3 and Phe12 in mF3 (6, 8). A similar analysis allowed the upfield shifts of the aromatic protons of Phe12, in particular Hζ, to be linked to a ring current originating from His21.

Discussion Zinc finger transcription factors evoke intense interest at several levels. Besides the concern for understanding the structural basis of sequence specific DNA binding, the modest affinity of Zn2+ for some fingers such as TFIIIA and MTF-1 and the potential that the metal center is a site of reaction for toxic metal ions have attracted attention (19-23, 47-50). The experiments described here initially grew out of the finding that the selective binding of the nine Zn finger protein, Zn-TFIIIA, to the ICR of the 5S rRNA could be abrogated by exposure of either the free or the DNA-bound protein to Cd2+ (24,+ 25). The possible complexity of the interaction of xeno-

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biotic metal ions with a multifinger protein led to the present studies on a single finger peptide F3 of TFIIIA. Zn-F3 of TFIIIA specifically binds to its cognate DNA sequence as shown by EMSA experiments as in Figure 1 (40). This fact provides the validation for an inquiry into the properties of Zn-F3 and Cd-F3. The affinity of Zn-F3 for C-block DNA as measured by a dissociation constant of 2.6 × 10-5 is about four orders of magnitude lower than that of holo-TFIIIA or of the first three zinc fingers of TFIIIA (10, 51). The linker sequences between the zinc finger domains have a significant, positive effect on the specific DNA recognition (52, 53). Also, the fingerfinger contacts in adjacent zinc fingers may stabilize the complex with DNA (6, 54). One may consider the ∆G° of binding of Zn fingers 1-3 to the C-block (-49 kJ/mol, ref 10) as comprised of free energy changes due to the independent binding of each Zn finger to the DNA fragment plus the contributions from interactions between fingers that accompany binding described above and a significantly favorable chelate effect because all of the fingers are part of the same molecule. The sum of the free energies from the binding of three Zn-F3 molecules (-81 kJ/mol) would be sufficient to account for the affinity of Zn-F(1-3). Interestingly, if this provides a good estimate for independent Zn finger binding, then some of the interfinger interactions must provide a positive contribution to the overall free energy change. In the presence of Cd2+, inhibition of TFIIIA binding to DNA has been observed (24, 25). An apparent decrease in the binding affinity to C-block DNA was observed for Cd-F3 relative to Zn-F3 in the EMSA (Figure 1). Nevertheless, the substantial affinity of Cd-F3 for the C-block indicates that the substitution of Zn2+ with Cd2+ does not grossly disturb the conformation of Zn-F3. In contrast, Pb-F3 does not adopt the characteristic ββR conformation (25). The small impact of Cd2+ substitution for Zn2+ in F3 places F3 between Sp1 and tramtrak in the effect of Cd2+ on a series of C2H2 zinc fingers. It seems not to alter the quantitative binding of Sp1 to its cognate DNA and, thus, may not perturb its structure (27). In contrast, Cdtramtrak displays at least a 10-fold reduction in binding affinity for its DNA partner as well as a readily discernible loss of helical character associated with its DNA recognition helix (26). Finally, Cd2+ substitution destroys specific binding of TFIIIA to the ICR DNA (24, 25). Thus, it appears that the resultant effect of exchange of Zn2+ for Cd2+ upon zinc finger structure is dependent upon the specific structure under consideration. The differences in the strength of the interaction of ZnF3 and Cd-F3 with the C-block DNA, although small, may explain the loss in DNA binding of a multifinger protein like Zn-TFIIIA upon exposure to Cd2+. On the basis of the analysis above for Zn fingers 1-3, a conversion to Cd fingers 1-3 would decrease their independent binding affinity of each finger for the C-block by about 14 kJ/mol or 100-fold in the dissociation constant. This is likely to account for the observed loss in affinity of Cdexposed TFIIIA for the ICR. Nevertheless, the formation of alternative structures in Cd-TFIIIA remains a viable possibility as an explanation for its lack of association with the ICR (25). For example, the interaction of Cd2+ with multiple Zn finger sites might give rise to the formation of interfinger, Cd-C3H, and Cd-C4 complexes.

Krepkiy et al.

The formation of alternative metal binding sites that involve two fingers was not operative with Zn- or Cd-F3; data on the back-titration of the Co-mF3 with Zn2+ or Cd2+ indicated that a 1:1 ratio of the metal ion to peptide was present in the metal reconstituted peptides. Furthermore, the titration of apo-F3 and apo-mF3 with Cd2+ or Zn2+ as observed by 1D 1H NMR spectroscopy also demonstrated the formation of Zn or Cd species with the ratio of metal/peptide equal to 1:1 (data not shown). Finally, the coupling of 113Cd2+ to the protons of the cysteines and histidines of mF3 was indicative of the binding of Cd2+ to both cysteine and histidine residues as is the case in single finger structures (Figure 6). At the outset, it was envisioned that a comparison of the 3D conformations of Zn-F3 and Cd-F3 would provide an explanation for the deletarious effect of Cd2+ on TFIIIA binding. However, it was not possible to obtain sufficient NMR information to solve these structures because both 1D and 2D 1H NMR spectra were broadened, indicating that an ensemble of interconverting species described the structure of each molecule. Excess Cd2+ or Zn2+ did not change the spectra, demonstrating that the observed conformational flexibility corresponds to a property of these metal-peptide complexes, not to the presence of equilibria between free and bound free metal ions and peptide. Despite the conformational flexibility, both Zn-F3 and Cd-F3 were able to bind selectively to the C-block DNA, suggesting that in the association with the oligonucleotide a particular peptide conformation from an ensemble was stabilized. The observed conformational flexibility may be due to the presence of four residues between the histidine ligands (His-X4-His). For example, the yeast Zn finger peptide ADR1a, which includes this distribution of histidines ligands, exists as an equilibrium of two conformations (55). To accommodate the presence of four intervening amino acids instead of three, which would offer a more favorable metal binding geometry for the histidine side chains, Liao et al. showed that the helix between the two histidine ligands in F3 of zinc fingers 1-3 of TFIIIA is distorted by zinc coordination (11). That distortion may lower the effective binding affinity of the finger for Zn2+. Thus, it was hypothesized that the modest stability of Zn- and Cd-F3, as indicated by their relatively large metal ion dissociation constants, was consistent with the presence of multiple forms of the metallo peptides and that by modifying the sequence to reduce the dissociation constants, stable structures would result. The metal ion dissociation constants of Zn finger peptides display a remarkable range of 10-8 to 10-11 M (20, 25, 38). The peptide with the smallest Zn2+ dissociation constant is a consensus sequence peptide, CP-1, which has a smaller number of interligand amino acids, C-AAm-C and H-AAn-H, than does F3 (38). Whereas for CP-1, m ) 2 and n ) 3, m ) n ) 4 for F3. It was hypothesized that the difference in the Zn2+ binding constants among Zn finger peptides may correlate with the number of amino acids between the histidines and the cysteines. In particular, as discussed above, because both imidazole ligands are part of the finger helical region, a change in the number of interhistidine amino acids would formally alter the positioning of the ligands around the helix. In fact, the single zinc finger domains of CP-1 and Sp1 fingers 2 and 3 include three amino acids between the histidines. Each adopts a stable conformation, which has been solved by NMR spectroscopy (5, 7,

Interaction of Cd2+ with Transcription Factor IIIA

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38). Therefore, the mF3 peptide was constructed so that it had the same number and sequence of amino acids between the cysteine and the histidine residues as CP-1 (38). Surprisingly, the dissociation constants of the Zn2+ and Cd2+ complexes of F3 and mF3 were similar, despite the more favorable orientation of the imidazole ligands in the mF3 species. Nevertheless, the NMR behavior of the metal complexes of F3 and mF3 diverged. In contrast to the metallo-F3 complexes, the Zn- and Cd-mF3 structures that were stable on the NMR time scale could be solved. The obvious result of the NMR study was to demonstrate that the conformations of Zn-mF3 and Cd-mF3 are very similar (Figures 7 and 8). Indeed, apart from a modest difference in the conformational details of the helix in the two structures, it was not possible to point to obvious conformational differences that might reveal the basis of the effect of Cd2+ on TFIIIA binding with the ICR or on F3 binding to the C-block. In addition, however, the NMR spectra of Zn- and Cd-F3 indicate that their structures are flexible and might be described by a set of equilibria, in which Zn-F3′ is the conformation that binds to the C-block DNA:

Zn-F3: Zn-F3′ a Zn-F3′′ a Zn-F3′′′...

(4)

So, when Zn-F3 binds to its cognate DNA binding site,

Zn-F3 + DNA a Zn-F3′ ‚ DNA

(5)

The ∆G° for the reaction may be thought of as a sum of contributions from DNA binding by the conformational form that actually binds to DNA (e.g., Zn-F3′) and the unfavorable energy change related to stabilization of one member of the ensemble by DNA:

∆G° ) ∆GDNA binding° + ∆Gconformational stabilization° (6) Cd-F3 also exists as a manifold of related structures:

Cd-F3 ) Cd-F3′ a Cd-F3′′ a Cd-F3′′′...

(7)

Considering that the structure of Cd-mF3 stabilized at 278 K and that of Zn-mF3 observed at 298 K are very similar, we hypothesize that by analogy Zn-F3′ and Cd-F3′ also have the same structure and that the ∆GDNAbinding° is the same for each peptide. According to the studies with mF3, the Cd structure undergoes more temperature-dependent exchange between conformational forms than Zn-mF3. So, it is plausible that ∆Gconformational stabilization° is more positive for Cd-F3, in comparison with Zn-F3, thereby, reducing its overall ∆G° for binding and explaining the lower binding affinity of Cd-F3 for the C-block in comparison with Zn-F3. Although this argument may rationalize the different binding affinities of Zn- and Cd-F3 for the C-block DNA, it does not address the underlying basis for the subtly different conformations and conformational stabilities of the two peptides. Because the effect of the metal ion substitution is subtle, it is not currently possible to specify how the presence of Cd2+ causes minor perturbations in the structure.

Acknowledgment. We acknowledge the support of NIH Grants ES-04026 and ES-04184 and NSF instrumentation Grant NSF-9512622.

Supporting Information Available: Chemical shift and NOE information for Zn-mF3 and Cd-mF3. This material is available free of charge via the Internet at http://pubs.acs.org.

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