From the School of Molecular and Microbial Biosciences, G08, University of Sydney, New South Wales 2006, Australia
Received for publication, October 31, 2002 , and in revised form, May 2, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The majority of classical ZnFs are found in transcription factors, and a single transcription factor may contain more than 30 ZnF domains. It is well established that many of these domains mediate specific protein-DNA interactions (2), although recent reports have also demonstrated that at least some classical ZnFs can act as protein recognition motifs (for a review, see Ref. 3). The three-dimensional structures of a number of ZnF-DNA complexes have been determined, and a great deal is understood about the roles of individual amino acids in determining DNA sequence specificity (4, 5). These ZnFs bind to DNA using a tandem array of more than one ZnF (often three), where each ZnF contacts three base pairs.
The identification of ZnF motifs in the ever growing number of protein sequences is based primarily on the presence of conserved cysteine or histidine residues and the spacing between them. Further, DNA binding ability is sometimes inferred when three or more contiguous classical ZnFs are identified. This ability to infer function from sequence is becoming increasingly important as the amount of available sequence data increases. Interestingly, an examination of sequence data bases reveals that there are a number of proteins that contain sequences that correspond to one or more classical ZnFs, with the exception that the final zinc-ligating residue in the last ZnF is neither cysteine nor histidine (for examples, see Fig. 1). The question therefore arises as to whether these sequences are capable of folding and forming modules that are functional even in the absence of the typical final zinc-binding residue or whether the mutation leads to proteins that can no longer fold or (for example) bind to DNA.
|
In order to address this question, we have investigated the physical and
functional properties of a panel of point mutants based on the transcriptional
repressor basic Krüppel-like factor/Krüppel-like factor 3 (BKLF)
(6). BKLF binds to DNA
sequences containing CACCC motifs by means of three characteristic
Krüppel-like ZnFs (Krüppel-like fingers are a subset of classical
CCHH fingers with significant homology to those found in the archetypal
protein Drosophila regulatory protein Krüppel). We chose to
study the third or C-terminal zinc finger of BKLF (BKLF-F3, or BF3). We show
that the third zinc finger is essential for high affinity DNA binding and that
the final zinc-ligating histidine of BKLF-F3 can be substituted with a number
of different residues without severely compromising the DNA binding ability of
BKLF. Further, the mutant BF3 domains still bind Zn(II) and form substantial
secondary structure, although they are clearly not as well ordered as the
wild-type domain. Remarkably, the His Asn mutant binds Zn(II) with an
affinity that is essentially indistinguishable from that of the wild-type
BF3.
These results demonstrate that three side chains can be sufficient to bind a Zn(II) ion. Further, our data show that such domains, even when partially folded, can act in concert with other ZnFs to bind DNA. These findings further our understanding of the basic ZnF scaffold and show that attempts to identify DNA-binding ZnFs from amino acid sequence data should not necessarily exclude apparently incomplete ZnF configurations.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrophoretic Mobility Shift Assays (EMSAs)In order to
compare the effects of the point mutations, EMSAs were carried out. Reactions
were set up in a total volume of 30 µl, comprising 0.1 pg of
32P-labeled probe, 100 ng of recombinant protein, 10
mM Hepes, pH 7.8, 50 mM KCl, 5 mM
MgCl2, 1 mM EDTA, and 5% glycerol. The reactions used in
the quantitative EMSAs further included 1 mg/ml dI-dC, 5 mg/ml bovine serum
albumin, and 0.5% (v/v) Nonidet P-40. In these reactions, 67 pM
32P-labeled DNA and 02000 nM wild-type BF3 or
04500 nM mutant BF3A protein were used. After incubation on
ice for 10 min, the samples were loaded onto a 6% native polyacrylamide gel
made up in 0.5x TBE. The gel was then subjected to electrophoresis at 15
V/cm and 4 °C for 2 h, dried, analyzed, and quantified when necessary
using a PhosphorImager (Amersham Biosciences). The probes used in the
experiments were end-labeled according to standard procedures using
polynucleotide kinase (7). The
sequence was 5'-TAGAGCCACACCCTGGTAAG-3' (only the top strand is
shown). The DNA-binding affinities of the wild-type BF3 and mutant BF3A were
estimated by nonlinear least squares analysis as follows. The fraction of DNA
complexed to the BF1-3 proteins (fcx) was
calculated by using the equation fcx =
Icx/(Icx +
If), where Icx and
If are the intensities of the bands corresponding
to peptide-complexed DNA and free DNA, respectively. The DNA-binding affinity,
Ka, was obtained by fitting the experimentally
derived fcx to the equations,
![]() | (Eq. 1) |
![]() | (Eq. 2) |
Overexpression and Purification of BF3 and MutantsThe
wild-type and mutant BF3 proteins were expressed and purified in the same
manner. Luria broth was inoculated with transformed E. coli cells at
37 °C. When the A600 reached 0.6, protein
expression was induced with the addition of
isopropyl-
-D-thiogalactoside (0.4 mM). After 4 h,
the cells were pelleted by centrifugation and stored at 20 °C prior
to lysis. The cells were resuspended in lysis buffer (50 mM Tris,
50 mM NaCl, 1% Triton X-100, 1.4 mM phenylmethylsulfonyl
fluoride, 1.4 mM
-mercaptoethanol, pH 8.0) and lysed by
gentle sonication. The soluble fraction, separated from the insoluble fraction
by centrifugation (15,000 rpm, 4 °C, 20 min), was loaded onto
glutathione-Sepharose beads. Unbound proteins were washed away from the beads
with wash buffer (50 mM Tris, 100 mM NaCl, 10% (v/v)
glycerol, 1.4 mM phenylmethylsulfonyl fluoride, 1.4 mM
-mercaptoethanol, pH 8.0), and the beads were equilibrated with thrombin
buffer (50 mM Tris, 150 mM NaCl, 2.5 mM
CaCl2, pH 8.0). Thrombin was added, and the mixture was incubated
for either 2 h at 37 °C or overnight at 25 °C. The eluted peptides
were lyophilized, redissolved in water, and further purified by reversed-phase
HPLC (using a gradient of 595% acetonitrile in 0.1% trifluoroacetic
acid). The purified peptides were lyophilized and stored at 20 °C.
The identity of each peptide was confirmed using positive ion electrospray
mass spectrometry.
Far UV CD SpectropolarimetryEach HPLC-purified peptide was
dissolved in a solution containing TCEP (1 mM) and ZnSO4
(1 mM) to concentrations of either 30 µM (BF3, BF3D,
BF3E, and BF3N) or 12 µM (BF3A). The pH of these solutions was
2.0. A far UV CD spectrum of each peptide was taken, the pH was then
adjusted to
5.5 with the addition of NaOH, and a second spectrum of each
peptide was recorded. CD spectra were recorded at 25 °C on a Jasco J-720
spectropolarimeter equipped with a Neslab RTE-111 temperature controller.
Spectra were recorded in a 1-mm path length cell with a resolution of 0.5 nm
and bandwidth of 1 nm over the wavelength range of 190250 nm. Each
spectrum represented the average of three scans accumulated at a speed of 20
nm min1 with a response time of 1 s.
For the Zn(II) titration experiments, aliquots of a solution containing ZnCl2 (1320 mM; pH 5.5) were added to solutions of the wild-type BF3 (13 µM) and the mutant BF3N (20 µM), each containing 0.5 mM TCEP, pH 5.5. CD spectra were taken at each point in the titration, allowing 5 min for equilibration after each Zn(II) addition. Spectra were recorded over the wavelength range of 195200 nm with a resolution of 1 nm and as the average of 50 scans. Spectra were base line-corrected by subtraction of a spectrum of TCEP/ZnSO4 buffer alone.
Values of the association constant for zinc binding
(Ka) were determined by plotting the change in
ellipticity at a single wavelength against the total Zn(II) concentration.
Nonlinear least squares analysis was used to determine the Zn(II)-binding
affinities, using the equations,
![]() | (Eq. 3) |
![]() | (Eq. 4) |
Analytical UltracentrifugationSedimentation equilibrium experiments were performed using an OptimaTM XL-A analytical ultracentrifuge (Beckman Instruments) equipped with an An-60ti rotor. BF3E (at concentrations of 8.6, 20, and 33.6 µM; all in 1 mM TCEP, 1 mM ZnSO4, pH 5.6) was centrifuged against a matched buffer at 25 °C at 30,000 and 42,000 rpm, using double sector cells. Data were collected as A230 versus radius scans in 0.001-cm increments, and 10 scans were averaged for each data set. Base line correction was achieved by the subtraction of data recorded at 360 nm. Scans were taken at 3-h intervals and compared to ensure that the samples reached equilibrium. Analysis of the data was carried out using the NONLIN software (8), and the final parameters were determined by a nonlinear least squares fit of the data to a single species model. The goodness of fit was determined by examination of the residuals derived from the fit. The partial specific volume was determined from the amino acid sequence (9), and the solvent density was determined to be 0.997 g ml1 using the program SEDNTERP (10).
Nuclear Magnetic Resonance SpectroscopySamples of BF3 were prepared by dissolving either 3.5 mg of 15N-labeled BF3 or 11 mg of BF3 in H2O/D2O (95:5) containing 1.5 molar equivalents of both TCEP and ZnSO4. The pH was adjusted to 5.5 using 0.1 and 0.01 M NaOH; this gave sample concentrations of 1 mM for 15N-labeled BF3 and 3 mM for BF3.
For the comparison of wild-type BF3 with BF3E, both peptides were dissolved
in a solution containing TCEP (1 mM) and ZnSO4 (1
mM). The pH was adjusted to 5.5, and each solution was supplemented
with D2O (5%, v/v) and
d4-(trimethylsilyl)propionic acid (1 µl). The final
sample concentrations were 100 µM (BF3) and 85 µM
(BF3E). The pH of the BF3E sample was subsequently dropped to pH 2 to
obtain the spectrum of the unfolded protein. NMR spectra were recorded on a
Bruker DRX600 spectrometer equipped with a triple resonance (HCN) probe and
three-axis pulsed-field gradients. NMR spectra used for the comparison of BF3
and BF3E were recorded at 298 K, whereas those used for structure
determination were acquired at 280 K. The solvent signal was suppressed using
pulsed field gradients. One-dimensional 1H spectra consisted of 128
scans collected as 8,192 complex data points over a spectral width of 7,200
Hz. The following homonuclear two-dimensional experiments were recorded on the
unlabeled BF3 sample: TOCSY
(11) (
m = 70
ms), DQFCOSY (12), and NOESY
(13) (
m = 200
ms). Three-dimensional HNHA
(14) and three-dimensional
TOCSY-HSQC (15)
(
m = 70 ms) experiments were used to assign the
15N,1H HSQC spectra of the 15N-labeled BF3,
whereas the HNHA spectrum was used to derive
3JHNH
coupling constants.
Spectra were processed as described previously
(16). The 1H
frequency scale was referenced to
d4-(trimethylsilyl)propionic acid at 0.00 ppm.
Determination of the Structure of BF3Resonance assignment
was carried out using the standard homonuclear sequential assignment method
(17). Cross-peaks in the
two-dimensional NOESY spectra were integrated in XEASY and converted to upper
distance limits using the CALIBA module of DYANA
(18) and the default DYANA
parameters, except that an empirical correction of 0.5 Å was added to
the upper distance limits involving -methylene groups to account for
spin diffusion. Dihedral angle restraints for
angles were derived from
3JHNH
coupling constants
measured from the HNHA spectrum
(14). Residues with positive
angles were verified using methods as described in Ref.
19.
Initially, structure calculations were performed in DYANA, and additional
NOEs were assigned iteratively based on earlier sets of structures. At this
stage, structures were calculated without incorporating the Zn(II) atom.
Analysis of preliminary structures established that the N2
atoms of both histidine residues were coordinating the Zn(II). This was later
confirmed using a 1H-15N HMQC experiment optimized to
detect J-couplings in histidine side chains
(20). In order to include
additional distance and angle constraints that maintain the tetrahedral
bonding geometry and appropriate bond lengths with the zinc ion
(21), as well as to
incorporate ambiguous restraints, subsequent structural refinement was
performed in CNS (22) using
the package ARIA (23,
24). Manually assigned NOEs in
combination with the remaining ambiguous NOEs were included in the ARIA
structure calculations, and the latter were iteratively assigned in an
automated manner.
Briefly, one cycle of 200 structures was followed by seven cycles of 20 structures each and a final cycle of 1000 structures. Manually assigned NOEs were included in iteration zero as soft restraints. In each cycle, the seven lowest energy structures from the previous iteration were used to extract additional NOE restraints with a tolerance of 0.02 ppm in the F1 dimension and 0.015 ppm in the F2 dimension. If a restraint was violated by more than a predefined target value in over 50% of the seven structures, it was discarded. The violation target value was progressively reduced from 1000 Å in iteration zero to 0.1 Å in iteration eight. 18 of 782 distance restraints were discarded in the final iteration due to these violations. Ambiguous distance restraints were treated as described previously (25), and the peak volume cut-off was gradually reduced from 1.01 in the first iteration to 0.80 in the last. The final assignments made by ARIA were checked and corrected manually where necessary. Calculations were carried out in the simplified all-hydrogen PARALLHDG5.2 force field with nonbonded interactions modeled by the PROLSQ force field (26); floating chirality assignment (27) was used for all methylene and isopropyl groups.
Finally, the 25 lowest energy structures were subjected to water refinement using the standard water refinement protocol supplied by ARIA1.2 (28). The structures were immersed in a 7-Å shell of water molecules and were subjected to a short molecular dynamics simulation taking into account the Lennard-Jones, van der Waals, and electrostatic interactions and based on a slightly modified OPLS force field (29). These water-refined structures were visualized and analyzed using the programs MOLMOL (30), PROCHECK (31), and WhatIf (32). The coordinates of BKLF-F3 have been deposited in the Protein Data Bank under the accession code 1P7A.
Atomic Absorption SpectrometryBF3 and BF3D samples used in far UV CD analyses were dialyzed against sodium acetate buffer (10 mM, pH 5.4) containing 1 mM dithiothreitol, in order to remove excess Zn(II). The resulting samples were rechecked by CD to confirm that they had remained folded. The Zn(II) content of BF3 (26 µM) and BF3D (19 µM) samples were determined on a Varian SpectrAA 10/20 flame emission spectrometer at 213.9 nm. Concentrations were determined by reference to a standard curve constructed using atomic absorption standard zinc(II) solution (1000 mg/liter in 0.5 mol/liter nitric acid; BDH), diluted with Milli-Q® water to final concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/liter.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Given the dogma that four ligating amino acid side chains are required for the formation of a classical zinc finger structure, the existence of these variants led us to ask whether such sequences can form functional zinc finger domains, or if instead they represent vestigial domains that no longer play a role in the function of the parent protein.
CCHX Mutants of BKLF Bind to DNAIn order to address this question, we made a series of single site mutations in the zinc finger region of the transcriptional repressor BKLF (6). At its C terminus, BKLF has three contiguous CCHH fingers (Fig. 1B); this three-finger array binds with high affinity and specificity to oligonucleotides containing the sequence CACCC (6). We generated a fusion protein encompassing the three fingers of murine BKLF (BKLF-F13 residues 254344) C-terminal to glutathione S-transferase (GST). Six mutants were also prepared that harbored substitutions of the final histidine ligand in the third finger, namely aspartate, glutamate, asparagine, glutamine, alanine, and arginine (Fig. 1B). These constructs were termed BF1-3X (where X represents Asp, Glu, Asn, Gln, Ala, and Arg). Aspartate is known to act as a zinc ligand in LIM domains (34), a class of zinc fingers in which two zinc atoms are bound. Glutamate, asparagine, and glutamine were chosen as residues with side chains that could conceivably act as ligands to Zn(II), and alanine and arginine were selected as negative controls. We initially also attempted to make a derivative containing a cysteine, but despite several attempts, we were not successful at generating this protein by normal methods in E. coli, since the protein appears to be cytotoxic; this mutant was therefore not pursued further.
The seven proteins (wild-type BF1-3 and the six mutants) were tested for
their ability to bind a typical CACCC box motif, namely the motif in the
-globin promoter (see "Experimental Procedures"). GST alone
and probe alone were also included as negative controls in this experiment
(Fig. 2A). Remarkably,
all of the point mutants retained near native DNA binding ability. This result
was unexpected, given the generally accepted view that four zinc ligands are
required for the formation of a folded zinc finger domain and the low
probability that either arginine or alanine could coordinate Zn(II). Three
C-terminal truncation mutants were also tested for their DNA binding ability.
The mutant with four amino acids truncated at the C terminus of finger 3
(BF1-3(HMLV)), including the second zinc-ligating histidine, was able
to bind DNA, albeit with a reduced binding affinity
(Fig. 2B). In
contrast, protein constructs either with 17 amino acids truncated
(BF1-2
) or comprising only fingers 1 and 2 (BF1-2) were not able to
bind DNA (Fig.
2B).
|
In order to quantify the effect of the point mutations on DNA binding,
quantitative EMSAs were carried out using the wild-type and the alanine mutant
BF1-3A (Fig. 2,
CE). The wild-type protein binds a typical CACCC
box site with a Ka of (2.5 ± 0.4) x
107 M1. Surprisingly, the His
Ala mutation had a relatively modest effect on DNA binding; the mutant BF1-3A
bound DNA with a Ka of (2.16 ± 0.22)
x 106 M1
(Fig. 2E).
The observation that even the BF1-3A and BF1-3(HMLV) mutants bind DNA is most simply explained by one of two possible models. First, it could be that BF3 folds normally in the context of the mutants, irrespective of whether a fourth zinc ligand is present at position 341 (using the numbering in Fig. 1B). Second, it is possible that the BF1-3 mutants are either partially folded or completely unfolded but that the formation of structure and DNA binding take place concomitantly. A number of recent reports demonstrate simultaneous folding and binding events (35, 36).
BKLF Finger 3 Is a Typical Classical ZnFThe first step that we took in order to delineate the effect of the mutations was to determine the structure of the wild-type BF3 using NMR spectroscopy. Resonance assignment from homonuclear NMR spectra was straightforward, and the structure calculations were carried out using ARIA (23). The 20 structures with lowest overall energies (from the final refinement in water) were used to represent the solution structure of BF3 (Fig. 3A). The structures display good covalent geometry, judging from the small deviations from ideal bond lengths and angles, and good nonbonded contacts, as shown by the low value of the mean Lennard-Jones potential (Table I). The atomic coordinates for this family of conformers have been deposited with the Protein Data Bank.
|
|
The overall topology of BF3 conforms to the expected fold of classical CCHH
zinc finger domains: two short strands of antiparallel -sheet strands
linked by a rubredoxin-like turn are followed by an
-helix that
contains the two Zn(II)-coordinating histidine residues. The short
-sheet encompasses residues 319321 and 326328, and
hydrogen bonds are formed that involve the backbone amide protons of
Phe319, Cys321, and Phe328 and the carboxyl
oxygens of Phe328, Arg326, and Phe319,
respectively. The
-turn linking these two strands has hydrogen bonds and
dihedral angles consistent with the rubredoxin-like turn that is commonly
found in zinc-binding domains (see, for example, Refs.
37 and
38). A positive
angle
is often found in the residue following the second zinc-ligating Cys, and we
confirmed its existence in BF3 using the methods described in Ref.
19. The
-helix runs
from residue Ser331 to Leu343, although both
d
N(i, i + 3) and
d
N(i, i + 4) NOEs are observed
for residues 339343, suggesting that the conformation of this region
lies somewhere between an
-helix and a 310 helix. This
phenomenon is also seen in the last 34 residues of the
-helix of
the third ZnF of Sp1 (39).
Overall, the structure of BF3 is very similar to other classical zinc fingers. For example, it overlays with the second ZnF of Zif268 with a root mean square deviation of 0.82 Å over the ordered backbone atoms (Fig. 3C) (40).
BF3 Mutants Form Secondary Structure in a Zn(II)-dependent
FashionHaving established that the BF1-3 point mutants were all
capable of DNA binding and that the wild-type BF3 domain formed a normal
classical zinc finger fold, we sought to determine whether the mutants were
able to bind Zn(II). The six mutants were also produced as single finger GST
fusion proteins (termed BF3X). These overexpressed GST-BF3X proteins were
subjected to glutathione-affinity chromatography, and thrombin was used to
release the BF3X domains. However, all six mutant proteins exhibited partial
cleavage by thrombin at secondary sites. Useable amounts of wild-type BF3, as
well as mutants BF3D, BF3E, BF3N, and BF3A but not mutants BF3R or BF3Q were
isolated by reverse phase HPLC. In order to ascertain whether the mutant
domains were able to bind Zn(II), far UV CD spectra were recorded. The spectra
of wild-type BF3 and each of the mutants at low pH (pH 2) and in the
presence of excess Zn(II) are typical of unstructured polypeptides.
Surprisingly, however, an increase of the pH to 5.5 resulted in noticeable
increases in the secondary structure content of all domains
(Fig. 4A, black
lines). This was manifested as a red shift of the minimum and, in the
case of BF3, by the presence of positive ellipticity at low wavelengths.
Spectra recorded at pH 5.5 in the absence of Zn(II) were similar to the low pH
spectra (data not shown).
|
To assess more quantitatively whether Zn(II) binding is affected by these point mutations, the Zn(II)-binding affinities of the wild-type BF3 and the BF3N mutant were determined. Zn(II) was titrated into solutions of these domains, and the change in ellipticity at wavelengths between 195 and 200 nm was recorded (Fig. 4B). Surprisingly, nonlinear least squares fitting of the titration data revealed that BF3 and BF3N bound Zn(II) with similar affinities ((4.6 ± 1.4) x 104 M1 and (1.1 ± 0.6) x 105 M1, respectively).
BF3E Is Monomeric in Solution and BF3D Binds Zn2+ in a 1:1 RatioIn order to determine the aggregation state of one of the BF3X mutants, BF3E was subjected to sedimentation equilibrium analysis. Concentration versus radial distance profiles (Fig. 5) were obtained at two different rotor speeds, and nonlinear least squares analysis using the software NONLIN (8) showed that the data fitted very well to an ideal single species model with a molecular mass of 3,500 ± 200 Da. This value is in good agreement with the theoretical mass of 3,638 Da for Zn-BF3E. The Zn(II)/protein ratios for wild-type BF3 and BF3D were determined by atomic absorption spectrometry; both were 1:1.
|
BF3X Mutants Are Partially Folded in SolutionThe one-dimensional 1H NMR spectrum of wild-type BF3 (Fig. 6A) contained sharp and well dispersed signals, which is indicative of a folded protein with a significant degree of tertiary structure. In contrast, the one-dimensional 1H NMR spectrum of the BF3E (Fig. 6B), while exhibiting a reasonable amount of chemical shift dispersion compared with the same protein at pH 2 (Fig. 6C), was much broader. Given that BF3E is monomeric in solution (see above), the broadness indicates that BF3E is undergoing substantial chemical exchange on the microsecond-millisecond time scale.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Zinc- and DNA-binding Properties of the MutantsThe
transcriptional repressor BKLF contains a tandem array of three classical CCHH
zinc fingers that bind with high affinity to CACCC-containing DNA sequences.
The affinity of this interaction (3 x 107
M1) is comparable with those reported for other
known three-ZnF constructs binding to their cognate DNA sequence; for example,
the three CCHH ZnFs of Sp1 bind DNA with a Ka of
7 x 107 M1
(41). We have shown here that
the third zinc finger of BKLF is capable of making a substantial contribution
to DNA binding even when the most C-terminal of its four normal zinc ligands
(His341 in Fig.
1B) is mutated. The DNA binding affinity of the
three-finger construct BF1-3 is only reduced 10-fold when the last
zinc-ligating His mutated is Ala. Indeed, even a truncated construct lacking
any residue at the fourth zinc-ligating position (BF1-3(HMLV))
retains the ability to bind DNA, demonstrating that the DNA binding
contributions made by this "broken" finger do not depend on the
presence of four zinc ligands. A combination of CD, atomic absorption,
UV-visible, and 1H NMR data indicates that the point mutants are
capable of binding one molar equivalent of Zn(II), thereby forming substantial
secondary structure. Surprisingly, the Zn(II)-binding affinity of the single
BF3 finger appears not to be affected by the H N mutation.
Sedimentation equilibrium data confirm that zinc binding is achieved by a
single protein molecule (rather than by, for example, two molecules, each
contributing two ligands).
Examination of the sequence of BF3 reveals that there are no other amino acids in the vicinity of His341 that could readily substitute for that residue as the fourth zinc ligand. Whereas it is conceivable that His333 could serve this role, such an arrangement would require a substantial structural rearrangement, and it is unlikely that the resulting structure would be capable of contributing to DNA binding. Taken together therefore, our data indicate that three ligands are sufficient (although not optimal) for Zn(II) binding in BKLF. This conclusion is supported by an assortment of previous reports. For example, a truncated C2H2 ZnF (that is missing its last zinc-ligating histidine) was found to coordinate Co(II) in a tetrahedral manner and with an affinity comparable with that of the intact ZnF (42). In a second study, it was observed that when one of the three classical ZnFs of Zif268 was mutated to a CCHA configuration, the protein retained its ability to bind DNA (43). Finally, Cook et al. (44) reported a mutant of the Saccharomyces cerevisiae transcriptional activator ADR1, in which the second histidine of the C-terminal CCHH finger (in a two-ZnF tandem array) was substituted to a tyrosine. The ability of this mutant to activate transcription was reduced but not abrogated, and it was postulated that three zinc chelators might be sufficient to bind Zn(II) and maintain the protein in its active form.
Conformation of the MutantsThe enhanced susceptibility of
these mutants to proteolysis in E. coli, however, suggests that the
mutants do not form compact tertiary structures. Both the CD and the
1H NMR data support this conclusion. Whereas considerable amounts
of secondary structure were formed upon the addition of Zn(II), CD spectra of
the mutants were still somewhat different from the wild-type spectrum. The
broad nature of the 1H NMR spectrum of BF3E indicates the existence
of interconverting conformers in a chemical exchange process. Given that the
zinc-binding affinity for the BF3 His Asn mutant is not significantly
different from that of the wild-type domain, it is likely that the chemical
exchange arises from loose packing of the mutants, much like the molten
globule state often discussed in the context of protein folding. However, this
partial formation of structure is obviously sufficient to allow the
recognition of DNA in the context of the three-zinc finger construct BF1-3,
and it is possible that the mutated third finger forms more regular structure
concomitantly with DNA binding. Alternatively, the NMR broadening may be a
consequence of a change in the kinetics of metal binding; distinguishing these
possibilities would be problematic.
The Implications for Other StudiesThe identification of ZnFs from genomic sequence data relies in part on the presence of predictably spaced cysteine and histidine residues. Whereas a number of variant CCHX zinc fingers are picked up by automated methods, it is possible that others are not, given the apparent plasticity of the requirements for a functional ZnF. Results such as those presented here may assume increasing importance as the use of sequence data alone to infer protein function becomes more prevalent. Thus, our results indicate that care should be taken before presuming that CCHX zinc fingers found in putative proteins are nonfunctional or vestigial.
These data also cast a question on the routine use of alanine substitution to create null ZnFs in functional studies. Because it had been assumed that the structure and, hence, function of a ZnF depends on the ligation of Zn(II) by four ligands, alanine-substituted ZnFs were generally expected to be nonfunctional. This study indicates that, at least in the context of some sequences, alanine substitution mutants may retain significant residual ZnF structure and activity.
The CCHH zinc finger fold is a common scaffold from which proteins with different DNA binding specificities have been generated. It is the simple structure and small number of residues required to structurally stabilize the domains that makes them particularly versatile and adaptable. The high number of CCHH zinc finger genes in eukaryotic genomes suggests that they may have evolved early in evolution, and an intermediate containing only three zinc ligands that exhibited suboptimal function may have played a role in their evolution. Another interesting possibility is that the presence of an amino acid other than histidine and cysteine as a zinc ligand or the absence of one ligand may constitute a means by which ZnF proteins could be regulated. Whereas the CCHX ZnFs may still recognize DNA, their greater susceptibility to proteolytic degradation might reduce their cellular half-life. This idea might also be pertinent in the design of novel ZnFs as a method in which the bioavailability of designed ZnFs might be controlled.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Australian Research Council.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Supported by Australian Postgraduate Awards.
An Australian Research Fellow. To whom correspondence should be addressed.
Tel.: 61-2-9351-3906; Fax: 61-2-9351-4726; E-mail:
j.mackay{at}mmb.usyd.edu.au.
1 The abbreviations used are: ZnF, zinc finger; EMSA, electrophoretic
mobility shift assay; TCEP, tris(2-carboxyethyl)phosphine; HPLC, high pressure
liquid chromatography; GST, glutathione S-transferase; BF3, BKLF-F3;
TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY,
NOE spectroscopy; HSQC, heteronuclear single quantum correlation
spectroscopy.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|