THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 31, pp. 21934 –21941, August 4, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Effects of Common Cancer Mutations on Stability and
DNA Binding of Full-length p53 Compared with
Isolated Core Domains*
Received for publication, May 2, 2006, and in revised form, June 5, 2006 Published, JBC Papers in Press, June 5, 2006, DOI 10.1074/jbc.M604209200
Hwee Ching Ang1, Andreas C. Joerger, Sebastian Mayer, and Alan R. Fersht2
From the Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom
The tumor suppressor protein p53 is a transcription factor that
plays a critical role in the network of signals that control the fate of
a cell (1). In about half of human tumors, p53 is inactivated as a
result of point missense mutations in the sequence-specific
DNA binding core domain of the protein (2, 3). Six “hot spots”
are most frequently associated with cancer: Arg175, Gly245,
Arg248, Arg249, Arg273, and Arg282 (3) (Fig. 1). From quantitative folding and DNA binding studies of numerous cancer-associated core domain mutants, three phenotypes have been
broadly categorized: (i) DNA contact mutations with only
minor effects on folding and stability, such as R273H; (ii) mutations that disrupt the local structure, mainly in proximity to the
DNA binding surface, which destabilize the protein by ⱕ2 kcal/
mol relative to wild type, such as G245S; and (iii) highly destabilizing mutations, such as R175H, which destabilize the protein by ⬎3 kcal/mol (4 – 6). The structural effects of the
* This
work was supported by the Medical Research Council and Cancer
Research UK. 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.
1
Supported by the Singapore Agency for Science, Technology and Research
(A*STAR).
2
To whom correspondence should be addressed: Centre for Protein Engineering, Medical Research Council, Hills Road, Cambridge CB2 2QH, UK.
Tel.: 44-1223-402-136; Fax: 44-1223-402-140; E-mail: [email protected].
21934 JOURNAL OF BIOLOGICAL CHEMISTRY
mutations R249S and R273H have recently been elucidated by
x-ray crystallography (7).
p53 has several domains: the N-terminal transactivation
domain (residues 1– 63) (8), the proline-rich regulatory domain
(residues 64 –92) (9, 10), the DNA binding core domain (residues 94 –312) (11), the tetramerization domain (residues 324 –
355) (12), and the C-terminal domain (residues 360 –393) (13).
The core domain and the C-terminal domain bind DNA. The
isolated core domain (p53C)3 binds specifically to a doublestranded DNA consensus site containing two copies of the
10-base pair “half-site” motif 5⬘-PuPuPuC(A/T)(T/A)GPyPyPy-3⬘ (Pu ⫽ A/G, Py ⫽ T/C) that can be separated by up to 13
bases (14). p53C monomers bind specific DNA to give a 4:1
complex (15, 16). Binding to a minimal tetrameric p53 construct comprising the core and tetramerization domains is
cooperative with a stoichiometry of two dimers per DNA molecule (16). The C-terminal domain, its post-translational modifications and its role as a regulatory domain have been extensively studied and debated (13, 17–24). This domain has been
shown to bind nucleic acid in a sequence-nonspecific manner
(13, 25) with a strong electrostatic component (26).
A new approach in cancer therapy is to use drugs that can
rescue the activity of mutant p53 (6, 27–29). To assess further the feasibility of rescue drug therapy, we have quantitatively analyzed the effect of p53 cancer mutations on the
stability of full-length protein and the effect of the contact
mutation R273H on p53-DNA interactions. We used an
engineered thermostable mutant of p53 core domain
(T-p53C), containing the mutations M133L/V203A/N239Y/
N268D (30, 31) to produce p53 mutant proteins suitable for
accurate biophysical measurements.
EXPERIMENTAL PROCEDURES
Protein Expression and Purification—T-p53C, T-p53CT,
and mutants of these constructs were purified as previously
described (16, 31). The plasmid for human p53 (residues
1–393) subcloned into the polylinker region of vector pET24a(⫹) (Novagen) using the NdeI and EcoRI restriction sites
was kindly provided by C. Blair. Additional point mutations
3
The abbreviations used are: p53C, p53 core domain (residues 94 –312);
T-p53C, thermostable variant of p53 core domain (residues 94 –312) containing the four point mutations M133L, V203A, N239Y, and N268D;
T-p53CT, thermostable p53 truncation mutant (residues 94 –360, comprising the core and tetramerization domains); T-p53FL, thermostable variant
of p53 full-length protein; DTT, dithiothreitol; KD, dissociation constant;
AUC, analytical ultracentrifugation; DSC, differential scanning calorimetry.
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Common cancer mutations of p53 tend either to lower the
stability or distort the core domain of the protein or weaken its
DNA binding affinity. We have previously analyzed in vitro the
effects of mutations on the core domain of p53. Here, we
extend those measurements to full-length p53, using either
the wild-type protein or a biologically active superstable construct that is more amenable to accurate biophysical measurements to assess the possibilities of rescuing different types
of mutations by anticancer drugs. The tetrameric full-length
proteins had similar apparent melting temperatures to those
of the individual domains, and the structural mutations lowered the melting temperature by similar amounts. The thermodynamic stability of tetrameric p53 is thus dictated by its
core domain. We determined that the common contact mutation R273H weakened binding to the gadd45 recognition
sequence by ⬃700 –1000 times. Many mutants that have lowered melting temperatures should be good drug targets,
although the common R273H mutant binds response elements too weakly for simple rescue.
Stability and DNA Binding Affinity of Full-length p53 Mutants
were introduced using the QuikChange site-directed
mutagenesis kit (Stratagene). The expression vector was transformed into Escherichia coli BL21 for overexpression. Expression cultures were incubated at 37 °C at 250 rpm until OD600
reached ⬃0.8. Zinc sulfate was added to give a final concentration of 100 ␮M, and protein expression was induced with 500
␮M isopropyl ␤-D-thiogalactoside. Cells were harvested 16 h
later by centrifugation. The cell pellet from 3 liters of culture
was suspended in cell-cracking buffer of 50 mM NaPi, pH 7.5,
150 mM NaCl, 5 mM DTT, and 15% glycerol and cracked using
an Emulsiflex C5 high pressure homogenizer (Glen Creston).
The soluble fraction was loaded onto a Heparin HP column and
eluted with a 0 –1 M NaCl gradient over 20 column volumes.
The pooled fractions from this column were diluted 10-fold
with 25 mM Tris/Bis-Tris propane buffer pH 9, 5 mM DTT, and
15% glycerol, loaded onto a Poros 20HQ anionic exchange column and eluted with a 0 –1 M NaCl gradient over 20 column
volumes. The pooled fractions were purified further on a Superdex 200 26/60 preparative gel filtration column (Amersham
Biosciences) in 25 mM NaPi, pH 7.2, 300 mM NaCl, 5 mM DTT,
and 10% glycerol. The purified p53 proteins were greater than
95% pure as judged by SDS/polyacrylamide gel. Protein samples
were flash-frozen and stored in liquid nitrogen for further use.
DNA Duplex Assembly and Purification—Double-stranded
DNAs were assembled from HPLC-purified oligonucleotides
AUGUST 4, 2006 • VOLUME 281 • NUMBER 31
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FIGURE 1. Wire frame model of p53 core domain bound to gadd45 consensus DNA (PDB ID code 1TSR, molecule B). Secondary structural elements are highlighted by semitransparent ribbons and cylinders. A ␤-sandwich provides the scaffold for the DNA binding surface, which is rich in basic
amino acids and contains a zinc ion (yellow sphere). The six residues that are
most frequently mutated in human cancer are shown in orange. The small
blue spheres indicate the location of the mutation sites in the superstable
quadruple mutant T-p53C. Trp146 (highlighted in green) was used as a fluorescence probe in this study. The figure was generated using MOLSCRIPT (46)
and RASTER3D (47).
and purified according to procedures already described (16, 32).
The oligonucleotides were labeled on the 5⬘-end of the forward strand with fluorescein. Only the forward DNA strand
was labeled to avoid energy transfer between fluorophores.
The gadd45 promoter sequence was verified against the
published recognition element (33), and encoded by the oligonucleotides 5⬘-fluorescein-GTACAGAACATGTCTAAGCATGCTgGGGAC and 5⬘-GTCCCcAGCATGCTTAGACATGTTCTGTAC. Bases in capital letters agree with the
published consensus sequence (14). Nonspecific DNA (random
DNA) that did not contain a p53 recognition element was
encoded by the oligonucleotides 5⬘-fluorescein-AATATGGTTTGAAATAAAGAGTAAAGATTTG-3⬘ and 5⬘-CAAATCTTTACTCTTTATTCAAACCATATT-3⬘.
Equilibrium Denaturation—Samples for urea denaturation
experiments were prepared using a Hamilton MicrolabM dispenser from stock solutions of urea, buffer, and protein to contain 1 ␮M protein in 25 mM sodium phosphate buffer, pH 7.2,
150 mM KCl, 5 mM DTT, and increasing concentrations of urea.
Samples were incubated at 10 °C for 14 h prior to measurement.
The intrinsic fluorescence spectra of p53, excited at 280 nm,
were recorded in the range of 300 – 400 nm on a PerkinElmer
Life Sciences LS50B spectrofluorometer equipped with a
Waters 2700 sample manager and controlled by laboratory
software. The data were analyzed as previously described (4).
Differential Scanning Calorimetry (DSC)—DSC experiments
were performed using a Microcal VP-Capillary DSC instrument (Microcal, Amherst, MA) with an active cell volume of
⬃125 ␮l. Temperatures from 10 to 85 °C were scanned at a rate
of 250 °C/h. Protein samples were buffer-exchanged into a
buffer of 25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 5 mM
DTT. This buffer was also used for baseline scans. For core
domain, 100 ␮M protein was used. For full-length protein, 15
␮M (in monomeric units) protein was used. A pressure of 2.5
bars (nitrogen) was applied to the cell. The data were analyzed
with ORIGIN software (Microcal). The average apparent Tm
value is presented in parentheses in Fig. 2.
Analytical Centrifugation—All analytical centrifugation
(AUC) experiments were performed at 10 °C using a Beckman Optima XL-I centrifuge and a 60Ti rotor. To determine
DNA binding affinities, samples of 5 ␮M 5⬘-fluorescein-labeled gadd45 DNA and 100 ␮M T-p53 monomers were made
up in 25 mM sodium phosphate, pH 7.2, 150 mM KCl, and 5
mM DTT. To measure the stoichiometry and dissociation
constant for protein binding to fluorescein-tagged DNA,
absorbance values at 495 nm and interference patterns at 675
nm were recorded, and the data were analyzed using UltraSpin software. The apparent dissociation constant per monomer was calculated as KD ⫽ ([free DNA]/[complex]) ⫻ [free
protein] and presented in Table 2. This value is equivalent to
P50.
Fluorescence Anisotropy—Fluorescence anisotropy measurements were recorded on a PerkinElmer Life Sciences LS55
Luminescence Spectrometer equipped with a Hamilton Microlab titrator and controlled by laboratory software. The excitation (␭ex) and emission (␭em) wavelengths used were 480 nm
and 530 nm, respectively, and the slit widths for excitation and
emission were 15 nm and 20 nm. The photomultiplier voltage
Stability and DNA Binding Affinity of Full-length p53 Mutants
TABLE 1
Changes in free energy on urea-induced unfolding of p53 core
domain mutants
Data were collected at 10 °C in 25 mM sodium phosphate, pH 7.2, 150 mM KCl,
5 mM DTT. The mean m value of 13 urea denaturation curves at 10 °C is 2.89
(⫾0.11) kcal mol⫺1 M ⫺1, which was used to calculate the difference between
关D兴50%
关D兴50%
wild type and mutant proteins, ⌬⌬GD-N
, from the equation, ⌬⌬GD-N
⫽
⬍m⬎ ⌬ 关D兴50%.
Mutation
V143A
R175H
G245S
R249S
R273H
R282W
a
b
关D兴50%
⌬⌬GD-N
T-p53C
Wild type (p53C)a
kcal/mol
kcal/mol
3.7 ⫾ 0.15
2.5 ⫾ 0.10
0.8 ⫾ 0.04
2.0 ⫾ 0.12b
0.1 ⫾ 0.02b
3.0 ⫾ 0.20
3.5 ⫾ 0.06
3.5 ⫾ 0.06
1.2 ⫾ 0.03
2.0 ⫾ 0.05
0.3 ⫾ 0.04
3.3 ⫾ 0.10
Data for mutations in the wild-type context are taken from Refs. 4 and 45.
Data are taken from Ref. 7.
RESULTS
Common Cancer Mutations Have a Similar Effect on the
Stability of T-p53C, Wild-type p53C, and Full-length p53
We measured the effects of structural hot spot mutations
(R175H, G245S, and R282W) and the classic temperaturesensitive mutation V143A on the stability of T-p53C by urea
21936 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 2. Stability measurements of T-p53 and hot spot variants. A, ureainduced unfolding of T-p53C and hot spot variants, represented as fraction of
unfolded protein versus concentration of denaturant. Unfolding was monitored by fluorescence, at 10 °C in 25 mM sodium phosphate, pH 7.2, 150 mM
KCl, 5 mM DTT. The main probe for denaturation is Trp146, which is located
away from the DNA binding surface, at the far end of the ␤-sandwich. B,
thermal denaturation curves are shown for core domain (T-p53C) and its hot
spot variants (lower set of 7 curves), and for the full-length protein (T-p53FL)
and its hot spot variants (upper set of 5 curves). Average apparent melting
temperatures (Tm) are given in parentheses. Representative melting curves
(raw data) are shown and are offset for clarity. The apparent Tm for wild-type
p53C and p53FL are 44.1 °C and 45.1 °C, respectively.
denaturation (Table 1 and Fig. 2A). Overall, the trends followed the wild-type protein (4, 5). G245S destabilized the
core domain by 1.2 kcal/mol in wild type and 0.8 kcal/mol in
T-p53C. The structural mutations V143A, R175H, and
R282W that substantially destabilize the wild type by 3.3–3.5
kcal/mol also caused major stability loss in T-p53C, ranging
from 2.5–3.7 kcal/mol.
In the full-length protein, the signal from Trp146, which was
used to monitor the unfolding of the core domain by urea denaturation, is masked by the relatively strong fluorescence signal
from 3 tryptophan residues in the natively unfolded N-terminal
region. We therefore studied the effects of mutations on the
stability of full-length p53 by differential scanning calorimetry
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used was 950 with an integration time of 5 s for each measurement. The concentrations of tetrameric constructs were 1–50
␮M (dimers). The initial concentrations of DNA were 4 –10 nM.
The experiments were performed at 10 °C in 25 mM imidazole, pH 7.2, 213.4 mM NaCl, 5 mM DTT, with a total ionic
strength of 225 mM, as described by Weinberg et al. (16) to
minimize artifacts caused by nonspecific binding effects. For
T-p53CT-R273H, experiments were performed in the presence of 10% glycerol to prevent aggregation. Control experiments comparing T-p53CT binding gadd45 in 0, 5, and 10%
glycerol showed minimal differences in binding affinities.
Proteins were buffer exchanged into the reaction buffer
using NAP-10 columns (Amersham Biosciences), and protein
concentrations were measured spectrophotometrically immediately prior to use using molar extinction coefficients calculated according to the method of Gill and von Hippel (48). p53
was titrated into a cuvette containing fluorescein-labeled DNA,
and the solution was stirred for 30 s. After 60 s, the fluorescence
and fluorescence polarization values were measured, using an
integration time of 5 s. Different data analyses were performed
depending on the protein concentrations used.
(i) At concentrations of p53 far below Ktd: When the affinity
for DNA is very high, the measurements are made at concentrations of p53 that are much below the dissociation constant
for tetramers into dimers (Ktd). It can be assumed that unbound
protein is dimeric under the reaction conditions and does not
tetramerize until bound to DNA. The binding isotherm simplifies to that of the simple sequential two-site model, which was
previously used to describe the sequence-specific DNA binding
of p53CT (15, 16).
(ii) At concentrations of p53 approaching or exceeding Ktd:
At the other extreme, for weak binding to DNA, measurements
are made at concentrations of p53 that approach or exceed the
value of Ktd. The concentration of tetramer at each titration
point was calculated and plotted to give the KDNA value directly.
Stability and DNA Binding Affinity of Full-length p53 Mutants
TABLE 2
Binding of monomeric p53 to specific gadd45 DNA, as measured by
analytical ultracentrifugation at total ionic strength of 210 mM
KD values were determined by AUC at 10 °C in 25 mM sodium phosphate, pH 7.2,
150 mM KCl, and 5 mM DTT, at a total ionic strength of 210 mM. Measurements
were performed with 5 ␮M 5⬘-fluorescein-labeled double-stranded DNA that contained the 20-base pair, specific recognition element from the gadd45 promoter,
and 100 ␮M protein. Data were analyzed as described under “Experimental Procedures.” Data (except for T-p53C-G245S) have been published (7).
Protein
KD
p53C
T-p53C
T-p53C-G245S
T-p53C-R249S
T-p53C-R273H
14 ⫾ 1
10 ⫾ 3
35 ⫾ 2
56 ⫾ 3
51 ⫾ 4
␮M
Binding of DNA to Full-length p53 and Shorter Constructs
We have previously reported the DNA binding affinities of
p53 core domains by analytical ultracentrifugation studies (7).
The affinity of T-p53C-G245S using the same technique under
similar conditions was 35 ⫾ 2 ␮M (Table 2).
We analyzed the binding to tetrameric p53 using two constructs to distinguish between specific DNA binding to the core
and nonspecific binding to the C terminus: (i) a minimal tetrameric construct consisting of the core and tetramerization
domains (T-p53CT), and (ii) the full-length protein (T-p53FL)
4
S. Mayer and A. R. Fersht, unpublished data.
AUGUST 4, 2006 • VOLUME 281 • NUMBER 31
FIGURE 3. Hill plots for the binding of T-p53CT and T-p53FL to gadd45
DNA. Binding was cooperative in each case. Hill coefficients for T-p53CT and
T-p53FL were 1.95 and 1.99, respectively.
FIGURE 4. p53 in a dimer-tetramer equilibrium. p53 can bind DNA as a
dimer or tetramer. Where p53 exists in a dimer-tetramer equilibrium over the
range of measurement of DNA affinity, this two-component model is applied,
ignoring the monomers.
(24, 26). The DNA binding experiments were conducted using
fluorescence anisotropy at an ionic strength of 225 mM to
reduce nonspecific electrostatic interactions at the DNA binding interface, which is positively charged and contains numerous arginine residues that are involved in DNA binding (16).
Two types of 5⬘-fluorescein-labeled 30-mer double-stranded
DNA were tested: one that contained the 20-base pair specific
recognition element from the gadd45 promoter (gadd45);
another that did not contain a p53 recognition element (random DNA) (16, 32).
We analyzed the binding data for T-p53CT and T-p53FL
using the Hill equation, as previously described (16) (Fig. 3).
The Hill coefficient for binding to gadd45 promoter DNA was
1.95 for T-p53CT and 1.99 for T-p53FL (Fig. 3), consistent with
previous results for p53CT binding p21 and Mdm2 promoter
DNA (16), and indicates a cooperative binding event for
T-p53CT and T-p53FL binding gadd45 promoter DNA.
DNA Binding of the Minimal Tetrameric Construct Comprising Core and Tetramerization Domains—Tetrameric
p53 is in a dynamic tetramer-dimer equilibrium (Fig. 4). The
equilibrium constant for the minimal tetrameric construct
dissociating into dimers is 400 nM (per p53 dimer at 10 °C)
(34). p53 mutants that bind DNA weakly are present as tetJOURNAL OF BIOLOGICAL CHEMISTRY
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(DSC). The observed Tm is not the true melting temperature
but an apparent one because p53 does not denature reversibly
with increasing temperature (5). Although the data are qualitative, they reflect the relative destabilizing effects of the mutations; the apparent Tm values of T-p53C paralleled the reversible urea-induced thermodynamic data (Fig. 2, A and B). The
apparent Tm values are also useful for assessing the stability of
p53 in vivo.4
The apparent Tm of T-p53C-R273H was virtually the same
as T-p53C at 49.9 °C. The apparent Tm of T-p53C-G245S
was reduced to 47.3 °C. The apparent Tm of T-p53C-R249S
was further reduced to 45.0 °C. The highly destabilizing mutations, R175H and V143A, greatly reduced the Tm to 42.5 °C and
43.0 °C, respectively. Experiments for full-length p53 were done
with ⬃4 ␮M protein. Because the equilibrium constant for fulllength p53 tetramers dissociating into dimers is 300 nM (per p53
dimer at 10 °C) (34), the protein should exist as tetramers at this
concentration. Control experiments (not shown here) showed
no large effects of concentration on aggregation, so the apparent Tm values were qualitative but relative as the observed values depend on heating rate.
The apparent Tm of wild-type full-length p53 was 45.1 °C,
close to that of wild-type core domain 44.1 °C. In full-length
T-p53FL, the cancer mutations G245S, R249S, and R273H
showed the same relative effect on stability as in core domain
T-p53C (Fig. 2B). Introduction of the R273H mutation had little effect on the stability, and the apparent Tm shifted marginally to 48.2 °C. The structural mutation G245S reduced the
apparent Tm from 49.7 °C to 47.3 °C. The mutation R249S,
which was more destabilizing than the G245S mutation, further
reduced the apparent Tm to 43.8 °C.
Stability and DNA Binding Affinity of Full-length p53 Mutants
ramers at the high protein concentrations used. Measurements of the concentration of p53 (in dimers) for 50% saturation (P50) for different constructs and mutants of p53 were
made. T-p53CT bound the gadd45 recognition sequence
with P50 of 21 ⫾ 2 nM (Table 3, Fig. 5A) and random DNA
about 30 times more weakly with P50 of 680 ⫾ 20 nM (Table
3 and Fig. 5B). In contrast, the minimal tetrameric T-p53CTR273H mutant bound gadd45 about 1000 times more weakly
than did T-p53CT (see “Discussion”). Nevertheless, it could
still discriminate between the two types of DNA used, and
bound gadd45 with P50 of 3000 ⫾ 400 nM, several times more
tightly than nonspecific random DNA (P50 of ⬎10,000 nM)
(Fig. 5, C and D).
DISCUSSION
Destabilizing Effects of Common Cancer Mutations in p53
Binding affinities are expressed in terms of P50 values. P50 is the protein concentraCore
Domain and Full-length Protein Follow the Same Trend—
tion (in dimers) at 50% saturation. The specific and nonspecific DNA used are
Common cancer mutations had the same relative effects on the
5⬘-fluorescein-tagged gadd45 and random DNA, respectively. The oligonucleotide
sequences are described under “Experimental Procedures.”
stability of wild-type p53 core domain and the stabilized core
P50
domain variant T-p53C. For example, R249S destabilizes both
Protein
Specific DNA
Nonspecific DNA
core domain variants by ⬃2 kcal/mol whereas the introduction of
nM
nM
the R273H mutation had virtually no effect on stability (7). The use
T-p53CT
21 ⫾ 2
680 ⫾ 200
of this stabilized T-p53 construct enabled us to produce full-length
T-p53CT-R273H
3000 ⫾ 400
⬎10,000
T-p53FL
4⫾2
360 ⫾ 60
p53 mutants for accurate biophysical measurements. Interest1500 ⫾ 200a
T-p53FL-R273H
1400 ⫾ 200a
ingly, all cancer hot spot mutations tested had the same relative
T-p53FL-G245S
55 ⫾ 7
250 ⫾ 50
T-p53FL-R249S
1200 ⫾ 200
850 ⫾ 100
effects on the stability of the full-length protein as measured in
a
Likely to be nonspecific binding to the C terminus.
isolated core domain (Fig. 8, A and B). This indicates that the core
domain is pivotal in governing the
overall stability of p53, and that all
mutation-induced stability changes
observed at core domain level directly
translate into similar relative stability
changes in the full-length protein.
Effects of R273H Mutation on Specific DNA Binding of p53—The
R273H contact mutation reduced
the binding of the gadd45 response
element by raising the P50 for the
wild-type minimal tetrameric core
and tetramerization domain construct from 21 to 3000 nM (Table 3).
The contact mutation did not completely abrogate DNA binding, consistent with previous mammalian
cell-based studies in which reduced
but detectable transactivation function is observed for the mutant
R273H (35–37) and mixed tetramers of the R273H mutant with 2 or
more wild-type monomers are transcriptionally active (38). These observations are also in good agreement with crystallographic studies,
FIGURE 5. DNA binding isotherms for minimal tetrameric T-p53CT and T-p53CT-R273H binding to 5ⴕ-fluorescein-labeled double-stranded 30-mers that contained either the recognition element from the which show that while a critical
gadd45 promoter or a randomly generated sequence of 30 bases (16, 32). A, T-p53CT binding gadd45 DNA; DNA contact is lost, the overall
B, T-p53CT binding random DNA; C, T-p53CT-R273H binding gadd45 DNA; D, T-p53CT-R273H binding random
DNA. Experiments were done at 10 °C in 25 mM imidazole, 213.4 mM sodium chloride (total ionic strength of 225 architecture of the DNA binding
surface is preserved (7).
mM), 5 mM DTT, and 10% glycerol.
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TABLE 3
Binding of tetrameric p53 to specific gadd45 DNA and nonspecific
random DNA, as measured by fluorescence anisotropy at a total
ionic strength of 225 mM
DNA Binding of the Full-length Proteins—The full-length
protein T-p53FL bound gadd45 DNA with P50 of 4 ⫾ 2 nM,
about 5 times more tightly than did T-p53CT (Table 3 and Fig.
6A). Like T-p53CT, T-p53FL also showed strong selectivity for
gadd45 over random DNA, and bound about 90 times more
tightly to gadd45 than random DNA (Table 3 and Fig. 6B). The
R273H mutation reduced the binding of gadd45 to the fulllength protein to P50 of 1400 ⫾ 200 nM (Table 3 and Fig. 6C).
But, this binding is mainly attributable to nonspecific binding
to the C terminus (24, 26), because the dissociation constant for
nonspecific DNA was 1500 ⫾ 200 nM (Table 3 and Fig. 6D). The
same is true for T-p53FL-R249S, which is a weakly binding
mutant (P50 of 1200 ⫾ 200 and 850 ⫾ 100 nM, respectively, for
binding to specific and nonspecific DNA) (Fig. 7).
Stability and DNA Binding Affinity of Full-length p53 Mutants
FIGURE 7. DNA binding isotherms for full-length mutants T-p53FL-G245S and T-p53FL-R249S binding to
5ⴕ-fluorescein-labeled double-stranded 30-mers that contained either the recognition element from
the gadd45 promoter or a randomly generated sequence of 30 bases (16, 32). A, T-p53FL-G245S binding
gadd45 DNA; B, T-p53FL-G245S binding random DNA; C, T-p53FL-R249S binding gadd45 DNA; D, T-p53FLR249S binding random DNA. Experiments were done at 10 °C in 25 mM imidazole, 213.4 mM sodium chloride
(total ionic strength of 225 mM), and 5 mM DTT.
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FIGURE 6. DNA binding isotherms for full-length T-p53FL and T-p53FL-R273H binding to 5ⴕ-fluoresceinlabeled double-stranded 30-mers that contained either the recognition element from the gadd45 promoter
or a randomly generated sequence of 30 bases (16, 32). A, T-p53FL binding gadd45 DNA; B, T-p53FL binding
random DNA; C, T-p53FL-R273H binding gadd45 DNA; D, T-p53FL-R273H binding random DNA. Experiments were
done at 10 °C in 25 mM imidazole, 213.4 mM sodium chloride (total ionic strength of 225 mM), and 5 mM DTT.
Comparing Binding to Monomeric, Dimeric, and Tetrameric
Constructs—There are various complications in attempting to compare
directly the binding of DNA to isolated core domains with oligomeric
p53 that is in equilibrium between
dimers and tetramers. One problem
is that as the dissociation constant
of tetrameric p53 into dimers is
about 400 nm, the binding of
response elements with affinities in
the nanomolar region are measured
in a p53 concentration range in
which it is mainly dimeric. Hence,
the Hill constant of close to 2 for the
binding of gadd45 DNA to fulllength constructs, showing that the
p53 dimers have effectively to associate to tetramers to bind to the
response elements that have 4 sites.
It may be shown that for tight binding response elements such as
gadd45 to wild-type proteins at concentrations ⬍⬍400 nM, the P50 value
is the square root of the product of
the dissociation constants of the tetramer into dimer (Ktd) and the DNA
from the tetramer (KDNA), i.e. P50 ⫽
公Ktd䡠KDNA (Fig. 4). For T-p53CT,
substituting the measured value of
the dissociation constant for the tetramer to dimer (400 nM) into the
equation, together with the P50
value obtained from DNA binding
experiments, gives a value of KDNA
for gadd45 and T-p53CT of 1 nM.
For weakly binding DNA-protein
complexes, such as T-p53CTR273H, we can obtain the true dissociation constant of DNA from the
tetramer, KDNA, by calculating the
amount of tetrameric p53 in solution using the 400 nM dissociation
constant value and plotting the
observed anisotropy against the tetrameric p53 concentration. The
KDNA for gadd45 and T-p53CTR273H was found to be 1000 ⫾ 200
nM. By comparing the KDNA values,
it can be seen that the intrinsic binding to the tetramer is weakened by
⬃1000 times on the mutation of
R273H.
The absolute values of binding of
DNA to tetrameric constructs cannot be quantitatively compared with
the binding to core domains
Stability and DNA Binding Affinity of Full-length p53 Mutants
because of differences in molecularity, which lead to vastly different entropies of binding. The effects of mutation can be compared, however. The P50 values for monomeric core domains
binding gadd45 are 10 ⫾ 3 ␮M for T-p53C and 51 ⫾ 4 ␮M for
T-p53C-R273H (Table 2) (7). At first sight, it seems that the
mutation of R273H in the monomeric core domain weakens
binding by only a factor of five. It can be shown that the
observed P50 for four monomers binding to DNA with four
binding sites is the fourth root of the product of the four individual dissociation constants for each binding event (⫽
(K1K2K3K4)1/4).5 The true relative binding affinity of “tetrameric” wild-type core domains (i.e. 4 domains to the four-site
response element) to the R273H mutant is, therefore,
(K1K2K3K4)R273H/(K1K2K3K4)wt, that is (51/10)4, ⬃700, and not
5.1, which is within experimental error of that calculated from
the tetrameric constructs.
Consequences of Mutations in Vivo—The similarity of the
thermodynamic stability of full-length p53 to that of core
domain and the parallel effects of destabilizing mutations indicate
that the studies on rescuing the stability of core domain provide a
valid framework for the design of drugs to rescue destabilized conformations. The rescue of destabilized mutants is a feasible drug
5
A. R. Fersht, unpublished data.
21940 JOURNAL OF BIOLOGICAL CHEMISTRY
Acknowledgments—We thank Dr. Dmitry Veprintsev for help with
analytical ultracentrifugation, Dr. Chris Johnson for help with DSC
experiments, Dr. Maria Rosario Fernandez-Fernandez and Dr.
Stacey Rutledge for helpful advice, and Caroline Blair for assistance
with protein purification. Wild-type full-length p53 was a kind gift
from Dr. Heather Peto.
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