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INTRODUCTION |
Human p53 plays an important role in tumor suppression (1-4). It
is a modular protein consisting of discrete functional domains, which
can be expressed and studied in isolation. In particular, residues
325-355 of human p53 (p53tet) spontaneously form a tetramer in
solution (see Fig. 1a) (5-7). Each monomer within the
context of the p53tet domain adopts an identical structure,
viz. a short N-terminal
-strand (residues 326-333)
followed by a turn and a C-terminal
-helical domain (residues
335-354). Two monomers associate in an antiparallel fashion through
contacts between
-sheet strands as well as hydrophobic interactions
involving
-helical residues to form a "primary dimer" (6, 7).
One significant salt bridge in the p53tet region occurs between
Arg337 of one subunit and Asp352 of its
adjacent subunit (side chain oxygen-nitrogen distance of 2.72 Å) (6),
stabilizing the structure of the primary dimer (8, 9). Two primary
dimers then self-associate through an interface derived from residues
located in their
-helical domains to form a "dimer of dimers,"
referred to as a p53 tetramer. Mutations of amino acids at this
interface have highlighted the importance of hydrophobic residues
leading to the formation of the tetramer as well as stable p53 dimers
(10-15). To date, knowledge relating to the contribution of charged
residues at this interface to the nature and stability of the tetramer
through ion pair formation remains minimal. In comparison, the impact
of ion pairs on the oligomeric state and stability of other
self-assembling peptide domains such as coiled-coil sequences is well
documented. Naturally occurring and engineered coiled-coil domains have
been shown to form homodimers as well as heterodimers (16-19) and
heterotetramers (20). Protein complexes such as the Fos-Jun heterodimer
(19), for example, occur as a result of charged groups in their
coiled-coil regions, which promote hetero-oligomerization through the
destabilization of homotypic interactions.
An examination of the crystal structure (6) of the human p53
tetramerization domain reveals the presence of one arginine (Arg342), one lysine (Lys351), and four
glutamates (Glu339, Glu343, Glu346,
and Glu349) within the boundaries of the dimer-dimer
interface (residues 338-351). Of these residues, the only pairs of
complementary charged side chains proximal enough to form an
intermonomer salt bridge involve Lys351 with
Glu343 and/or Glu346 (see Fig. 1b).
The side chain oxygen of Glu343 on one monomer was found to
be located 2.58 Å from the nitrogen side chain of Lys351
on another monomer. NMR structures of p53tet (7, 21) in solution have
revealed that Glu346 is apparently closer than
Glu343 to Lys351, although these ionic residues
are farther apart in these structures than in the crystal structure.
Finally, alignment of the tetramerization domain sequences of p53 from
Xenopus laevis (22) and rainbow trout (23) as
well as of human p73 and p63 (24-26) (see Fig. 1c)
indicates that the naturally occurring E343K mutation in their tetramerization domains is always coupled with a corresponding loss of
the positively charged lysine residue at position 351. Taken together,
these findings suggest that the presence of salt bridges involving
Lys351 with Glu343 and/or Glu346
would contribute four or more ionic interactions within the context of
the p53 tetramer interface, favoring the stabilization and self-association of primary dimers. To test this hypothesis, we engineered variants of the p53tet domain harboring charge-reversal mutations at positions 343, 346, and 351 (see Fig. 1c).
Mutants p53tet-E343K, p53tet-E346K, and p53tet-E343K/E346K will
result in dimer-dimer interfaces exhibiting a preponderance of
positively charged side chains, whereas mutant p53tet-K351E will
produce an interface enriched for negatively charged residues, both
scenarios leading to electrostatic interactions unfavorable for
tetramer formation. Conversely, the negative effects of introducing
like charges at the dimer-dimer interface should be nullified in the case of heterotetramers composed of dimers of p53tet-K351E paired with
p53tet-E343K, p53tet-E346K, or p53tet-E343K/E346K. The stability and
oligomeric state of these p53tet constructs were analyzed in a series
of biophysical experiments to resolve the role of such salt bridges at
the dimer-dimer interface.
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EXPERIMENTAL PROCEDURES |
Mutagenesis--
Plasmids pET-15b-p53-(310-360) and
pET-15b-p53-(310-360)-M340Q/L344R (15) were gifts from the laboratory
of Dr. Cheryl Arrowsmith (Ontario Cancer Institute). The plasmids
contain a synthetic gene coding for residues 310-360 of human p53
inserted into the NdeI and BamHI restriction
sites of the bacterial expression vector pET-15b (Novagen, Madison,
WI). The p53-(310-360) sequence is preceded by a vector-encoded
His6 metal ion affinity purification tag and a thrombin
cleavage site (see Fig. 1c). The E343K, E346K, and K351E
single mutants and the E343K/E346K double mutant of p53-(310-360) were
assembled by PCR mutagenesis following a two-step three-primer method
(27) using ProofStart DNA polymerase (QIAGEN, Mississauga, Ontario,
Canada). PCR products were purified from reaction mixtures or agarose
gels using QIAquick PCR purification and QIAquick gel extraction kits
(QIAGEN). The final PCR products were cloned into a pET-15b vector.
Mutations in the gene were confirmed by DNA sequencing. Plasmid
constructs were transformed into competent BL21(DE3) pLysS cells
(Novagen) according to standard methods (28).
Protein Expression and Purification--
Wild-type and mutant
His6-p53-(310-360) (i.e.
His6-p53tet) proteins were expressed and purified by the
same methods. Briefly, stocks of BL21(DE3) pLysS cells carrying the
appropriate plasmid were plated on LB-agar plates supplemented with 100 µg/ml carbenicillin and 34 µg/ml chloramphenicol. A single colony
was subsequently used to inoculate 40 ml of terrific broth
supplemented with the same antibiotics. The cultures were grown
overnight with shaking at 37 °C. A 15-ml aliquot of each culture was
used to inoculate 1.5 liters of preheated (37 °C) terrific
broth containing carbenicillin and chloramphenicol. The resulting
cultures were then grown at 37 °C with shaking until
A600 nm = 0.6~0.9 (2-3 h), at which point
0.5 mM isopropyl-
-D-thiogalactopyranoside
was added to the medium to induce protein expression. Cells were
harvested by centrifugation after a 3-h induction period.
Cell pellets (~7.5 g, wet weight) were subjected to three freeze/thaw
cycles and resuspended in ~3.7 volumes of buffer A (50 mM
Tris-HCl, pH 8.0, 500 mM NaCl, and 0.1% Triton X-100) with 20 mM imidazole, 1 mM phenylmethylsulfonyl
fluoride, 10 mM MgCl2, and 2.5 units/ml
Benzonase nuclease (Novagen). This suspension was placed on ice
and sonicated three successive times for 45 s, and the resulting
sonicate was centrifuged at 15,000 × g for 30 min. The
supernatant was loaded onto a 2.5-ml column of Talon metal affinity
resin (Clontech, Palo Alto, CA) equilibrated with buffer A containing 25 mM imidazole and 1 mM
phenylmethylsulfonyl fluoride, and the resin was washed with 25-50 ml
of the same buffer. Pure protein was eluted with 20 ml of buffer A
containing 200 mM imidazole. The eluate was dialyzed
extensively against 20 mM NH4HCO3,
and the protein was lyophilized and stored at
20 °C until used.
Purity was determined by SDS-PAGE with Coomassie staining (28)
and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)1 mass
spectrometry (Molecular Medicine Research Centre Mass Spectrometry Laboratory, University of Toronto).
Circular Dichroism Spectroscopy--
CD spectra were recorded on
an Aviv 62A DS circular dichroism spectrometer using a 0.5-cm path
length rectangular cuvette with a 2-ml sample volume. Protein samples
(10 µM) were prepared in sample buffer (25 mM
sodium phosphate, pH 7.0, and 100 mM NaCl). Wavelength
scans were recorded with a 1-nm spectral bandwidth (1 nm between
points) and an averaging time of 8 s. Ellipticity measurements at
222 nm were collected as a function of temperature for each p53tet
construct (or mixtures) using a 1-nm band width and a 50-s averaging
time. Measurements were recorded from 20 to 98 °C at 3 °C
intervals with a 1-min temperature pre-equilibration. Ellipticity
values were plotted as the fraction of unfolded protein versus temperature assuming a two-state folding model.
Ultracentrifugation--
Sedimentation equilibrium
ultracentrifugation experiments were performed on a Beckman Optima XL-I
analytical ultracentrifuge using an AN50-Ti rotor with six-channel
charcoal-Epon cells. Protein concentrations were 0.125, 0.25, and 0.5 mg/ml prepared in sample buffer as measured by UV spectroscopy
using the molar extinction coefficient for a free tyrosine residue
(
276 nm = 1450). Samples were centrifuged at 20 °C
at three different speeds for 24 h before equilibrium absorbance
measurements were taken at 230 nm. Association constants and molecular
masses were estimated using Beckman XL-I data analysis software in
which absorbance versus radial position data were fitted to
the sedimentation equilibrium equation using nonlinear least-squares
techniques (29).
Size-exclusion Chromatography--
Analytical size-exclusion
chromatography (SEC) experiments were performed on a Superdex-75HR
column (10 mm × 30 cm, Amersham Biosciences) operating at a flow
rate of 1 ml/min. Samples (0.8 mg in 400 µl) were injected onto the
column, and absorbance was monitored at 280 nm. The column was
calibrated with gel filtration standards from Bio-Rad.
Thrombin Cleavage of Mutant p53tet
Proteins--
His6-p53tet-E343K/E346K and
His6-p53tet-K351E were cleaved with thrombin using a
thrombin cleavage capture kit (Novagen). Two milligrams of each protein
were dissolved in 5 ml of 1× thrombin cleavage buffer (20 mM Tris-HCl, pH 8.4, 150 mM NaCl, and 25 mM CaCl2). Biotinylated thrombin (0.5 units,
0.25 units/mg) was then added to the reaction mixture, and the reaction
was left to proceed at room temperature for 16 h. The biotinylated
thrombin was subsequently removed with streptavidin-agarose, and the
cleaved His6 tag was eliminated with Talon metal affinity
resin. The filtrate containing pure cleaved p53tet-E343K/E346K or
p53tet-K351E, without the His6 tag, was dialyzed against 20 mM NH4HCO3, lyophilized, and stored at
20 °C until used. Samples were analyzed for cleavage and purity by SDS-PAGE. Complete cleavage was achieved, and cleavage at other sites in the protein did not occur, as no other low molecular mass
bands were detected.
Metal Affinity Experiments--
Lyophilized
His6-p53tet-WT, His6-p53tet-E343K,
His6-p53tet-E346K, His6-p53tet-E343K/E346K,
His6-p53tet-K351E, p53tet-E343K/E346K, and p53tet-K351E
were dissolved in sample buffer to final concentrations of 0.2 mM (1.6 mg/ml). Combinations of p53tet constructs (at a molar equivalence of each construct) were mixed and incubated at room
temperature for 1 h in microcentrifuge tubes. Fifty microliters of
Talon metal affinity resin were added to 300 µl of each mixture, and
the resulting samples were gently mixed at room temperature for 10 min.
The resin was pelleted by centrifugation (10,000 × g
for 30 s) and subsequently washed five times with 200 µl of sample buffer. Bound proteins were eluted in the presence of 100 µl
of sample buffer containing 0.5 M imidazole. Samples
corresponding to the original mixture of proteins prior to treatment
with Talon resin as well as aliquots of the supernatant after
incubation with Talon resin and of the eluate from the imidazole wash
were analyzed by SDS-PAGE.
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RESULTS |
Design and Structure of Human p53tet Constructs--
A wild-type
human p53-(310-360) construct with an N-terminal
His6 tag and a thrombin cleavage site as well as four
corresponding p53tet variants harboring mutation E343K, E346K,
E343K/E346K, or K351E (Fig.
1c) were expressed in
bacteria. The five 72-amino acid-long constructs were purified to
homogeneity by metal affinity chromatography (cobalt-based Talon
resin), and their masses were confirmed by MALDI-TOF mass spectrometry
and SDS-PAGE. The CD spectra of the p53tet-E343K/E346K and p53tet-K351E
analogs were similar to that of our p53tet-WT construct (data not
shown), suggesting a comparable secondary structure.

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Fig. 1.
Structure of the tetramerization domain of
human p53. a, three-dimensional structure of the
tetramerization domain of human p53 as determined by x-ray
crystallography (Protein Data Bank code 1c26) (6) using Swiss
PDB Viewer (available at www.expasy.ch/spdv) (35). One ionic network
involving Glu343, Glu346, and
Lys351 is depicted as a wire-frame model. b,
close-up view of the ionic network. Relevant distances between atoms
are indicated. c, sequences of the 72-amino acid-long p53tet
protein constructs used in this study. Vector-associated sequences
(histidine tag and thrombin cleavage site) are shown in
italics, and the p53tet minimum structural domain (residues
325-355 of human p53) is underlined. Glu343,
Glu346, and Lys351 are shown in
boldface. The site of thrombin cleavage is indicated by an
arrow. Alignment (performed with ClustalW) (36) of sequences
of the tetramerization domains of human p73 (24) and p63 (26) as well
as of p53 proteins from X. laevis (22) and
rainbow trout (23) is presented to highlight compensatory mutations
involving Glu343 and Lys351, which avoid
unfavorable charge effects.
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Oligomeric State of p53tet Mutants--
Wild-type and mutant
p53tet constructs were subjected to analytical ultracentrifugation at
20 °C to assess their oligomeric state. Representative results are
shown in Fig. 2. Data for all variants
were fitted to a single species; the apparent masses are listed in
Table I and indicate that p53tet-WT
(apparent mass of 31.8 kDa) is a tetramer in solution. The analog
p53tet-E343K/E346K is also predominantly a tetramer. However, its
apparent oligomeric state (3.5) is lower than that of p53tet-WT (3.9),
suggesting that the p53tet-E343K/E346K tetramer is less stable than its
p53tet-WT counterpart. The data for p53tet-WT and p53tet-E343K/E346K
can best be analyzed by assuming a monomer-tetramer equilibrium. The free energy change upon tetramerization (
G0)
for p53tet-WT was calculated to be
23.4 kcal/mol, which corresponds to a Kd (total protein concentration at which half
the protein exists as a tetramer) of 2.0 µM. This agrees
well with published values for similar p53tet peptides (30, 31). Using the same data treatment, the
G0 for tetramer
formation of p53tet-E343K/E346K was found to be
20.0
kcal/mol, which corresponds to a Kd of
13.3 µM. Thus, these calculations confirm the decreased
stability of p53tet-E343K/E346K relative to p53tet-WT. The apparent
oligomeric state of p53tet-K351E was calculated to be 2.0, indicating
that this mutant is a dimer in solution. Data were also fitted to a
monomer-dimer equilibrium; the Kd was calculated to
be very low (<10
15 M).

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Fig. 2.
Representative sedimentation equilibrium
ultracentrifugation data for p53tet variants, measured at 20 °C at
the indicated speeds. a, p53tet-K351E (33,000 rpm);
b, p53tet-E343K/E346K + p53tet-K351E (32,000 rpm).
Absorbance measurements were taken at 230 nm. The fit for a
is to a monomer-dimer equilibrium; the fit for b is a free
fit to a single oligomeric species. Residuals are multiplied by
103 for clarity.
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Table I
Apparent mass and oligomeric state of p53tet constructs derived from
sedimentation equilibrium ultracentrifugation studies
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The oligomeric state of the p53tet mutants was further confirmed by
SEC. Representative elution profiles are depicted in Fig. 3. Under these conditions, the tetrameric
p53tet-WT construct eluted at 10.3 min as a tetramer with an apparent
molecular mass of 44.3 kDa. As has been noted previously (10, 12), the
observed value is greater than the expected molecular mass for
tetrameric p53tet-WT because of the relatively unique nature of its
tetrameric structure. p53tet-M340Q/L344R, which is known to form dimers
(15), eluted at 11.5 min, establishing the retention time for a dimeric form of such constructs. p53tet-E343K, p53tet-E346K, and
p53tet-E343K/E346K (Fig. 3) eluted at times similar to p53tet-WT,
indicating that these mutants exist as tetramers. The analog
p53tet-K351E eluted at 11.3 min, pointing out that this construct, as
in the case of p53tet-M340Q/L344R, is a dimer. This finding supports
our analytical ultracentrifugation results (see above).

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Fig. 3.
Molecular size of p53tet variants.
Variants of human p53tet were analyzed by SEC. Injected samples (0.8 mg
in 400 µl) were dissolved in sample buffer, and absorbance was
recorded at 280 nm. Dashed lines indicate the
elution times of the p53tet-WT tetramer (t) and of the known
p53tet-M340Q/L344R dimer (d). The elution profiles of the
p53tet-E343K + p53tet-K351E and p53tet-E346K + p53tet-K351E mixtures
(not shown) are also similar to that of p53tet-WT.
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p53tet Constructs with Charge Inversions at Positions 351 and
343/346 Are Less Stable--
As shown above, the
sedimentation equilibrium ultracentrifugation data indicated that the
p53tet-E343K/E346K tetramer was less stable than p53tet-WT, with a
difference in
G0 for tetramerization of 3.4 kcal/mol. In addition, CD was used to measure the thermal stability of
wild-type and mutant forms of p53tet. All constructs displayed a
sigmoidal unfolding curve, which is indicative of cooperative
unfolding. Under the conditions of this study (25 mM sodium
phosphate, 100 mM NaCl, and 10 µM each p53
construct based on monomer concentration), p53tet-WT had a thermal
unfolding temperature (Tm) of 68 °C.
The effect of temperature on the fraction of folded structure as
calculated from changes in ellipticity at 222 nm (Fig.
4 and Table
II) suggested that the inversion of
charges at residues 343, 346, and 351 resulted in less stable
tetramers. The E343K and E346K mutations both posted lower
Tm values in relation to the p53tet-WT construct.
The E346K mutation had a greater effect on Tm
(60 °C) than the identical mutation at position 343 (E343K;
67 °C). When both E343K and E346K mutations were included, the
destabilizing effect was greater (Tm = 57 °C).
The K351E mutation to p53tet alone displayed the largest destabilizing effect (Tm = 53 °C), demonstrating an important
role for Lys351 in stabilizing the tetramer.

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Fig. 4.
Thermal stability of p53tet variants.
Temperature melting curves for individual p53tet constructs and various
combinations were determined by plotting ellipticity values derived
from CD measurements at 222 nm as the fraction of unfolded protein
(fu) versus temperature, assuming a
two-state folding model. Experiments were conducted with a protein
(monomer) concentration of 10 µM in sample buffer.
a, p53tet mutants compared with p53tet-WT. , p53tet-WT;
, p53tet-E343K; , p53tet-E346K; , p53tet-E343K/E346K; ,
p53tet-K351E. b, mixtures of p53tet mutants. , p53tet-WT;
, p53tet-E343K + p53tet-K351E; , p53tet-E346K + p53tet-K351E;
, p53tet-E343K/E346K + p53tet-K351E.
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p53tet-K351E and p53tet-E343K/E346K Specifically Form a
Heterotetramer--
Because p53tet-K351E forms a dimer in solution, it
can be used to study the formation of heterotetramers with other p53tet species. SEC results showed that when p53tet-K351E was mixed at equal
proportions with p53tet-E343K (data not shown), p53tet-E346K (data not
shown), or p53tet-E343K/E346K (Fig. 3), the result was a single peak
corresponding to a tetrameric species. This finding indicates that
p53tet-K351E associates with these mutants to form a 2:2
heterotetramer. This association is specific. For instance, p53tet-K351E did not associate with p53tet-WT, as demonstrated by the
elution profile of an equimolar mixture of these two proteins (Fig. 3).
Two peaks were observed for this mixture, with elution times very
similar to those of the individual p53tet components (10.5 min for the
wild-type tetramer and 11.2 min for the K351E dimer). The substantial
amount of dimeric species in the size-exclusion elution profile of the
mixture of p53tet-K351E and p53tet-WT indicated that an association of
p53tet-WT with p53tet-K351E did not occur.
Sedimentation equilibrium ultracentrifugation data (Fig. 2 and Table I)
also showed that an equimolar mixture of p53tet-E343K/E346K and
p53tet-K351E produced a new species that occurred as a tetramer (single-state free fit revealed an apparent oligomeric state of 3.9, identical to that of p53tet-WT). Importantly, both SEC and ultracentrifugation showed the absence of any dimeric species, suggesting that no uncomplexed p53tet-K351E remains in solution after
mixing. A fit of the ultracentrifugation data for the
p53tet-E343K/E346K + p53tet-K351E mixture to a monomer-tetramer
equilibrium revealed an apparent
G0 for
tetramerization of
23.5 kcal/mol, which corresponds to a Kd of 1.8 µM. Due to the complex
nature of the mixture (monomers, homodimers, homotetramers, and
heterotetramers could all potentially be present), the significance of
these values is open to question. However, the close correspondence
between the
G0 and Kd
values obtained and those observed for p53tet-WT (
G0 =
23.4 kcal/mol, Kd = 2.0 µM) suggests that the stability of the
heterotetramer is similar to that of p53tet-WT.
The temperature melting curves derived from CD spectra (Fig.
4b) precisely demonstrated that the increased stability
associated with this heterotypic interaction was much more dramatic
with the E343K/E346K double mutant than with either p53tet-E343K or p53tet-E346K alone. The temperature curves presented in Fig. 4 show
that when p53tet-E343K or p53tet-E346K was combined with p53tet-K351E,
the melting temperatures of the resulting mixtures (Table II) were
lowered relative to the Glu-to-Lys single mutant alone
(Tm = 66 °C for p53tet-E343K + p53tet-K351E
versus 67 °C for p53tet-E343K, Tm = 58 °C for p53tet-E346K + p53tet-K351E versus 60 °C for
p53tet-E346K). This finding indicates a lack of a stabilizing
interaction between these p53tet components. In contrast, Fig. 4 and
Table II show that when p53tet-E343K/E346K and p53tet-K351E were
combined, the result was a new species with a Tm of
66 °C. This temperature was dramatically higher than that of either
of the two mutants alone (57 °C for p53tet-E343K/E346K and 53 °C
for p53tet-K351E) and nearly equal to the value observed for p53tet-WT
(68 °C). These CD unfolding results support the sedimentation
equilibrium ultracentrifugation data, which also suggested that the
stability of the heterotetramer is similar to that of the p53tet-WT tetramer.
Selective Capture of His6-tagged p53tet
Complexes on a Metal Affinity Resin Demonstrates the Existence of
Heterotetramers--
The specific heterotypic association of
p53tet-E343K/E346K and p53tet-K351E was further assessed by monitoring
the binding of protein complexes composed of His6-tagged
and untagged p53tet constructs to cobalt-bound affinity resin
(Talon). The resulting band patterns observed by SDS-PAGE (Fig.
5) provide a unique signature describing
the nature of the complexes formed. Fig. 5a shows the results of combining together p53tet-E343K/E346K and p53tet-K351E. Lanes 1-3 clearly show that
His6-p53tet-E343K/E346K and His6-p53tet-K351E together specifically bound Talon resin (lane 2, no band),
but were eluted with a high concentration of imidazole (lane
3). As expected, neither of the p53tet constructs lacking the
His6 tag (lanes 4-6) was able to bind to the
metal affinity resin. (Protein bands were found in the wash fraction
(lane 5).) p53tet-E343K/E346K migrated slightly faster than
p53tet-K351E upon SDS-PAGE, such that
His6-p53tet-E343K/E346K ran at the same position as cleaved p53tet-K351E (lane 7). When this mixture was bound to the
affinity resin, both species bound to the resin (lane 8, no
band), and both species were eluted with imidazole (lane 9).
This finding demonstrates the specific association of these two
proteins to form a complex with affinity for metal ions. The reciprocal
combination yielded the same result, i.e. the association of
cleaved p53tet-E343K/E346K and His6-p53tet-K351E resulted
in a complex retained by the metal-bound resin (lanes
10-12). In this case, separate bands for the two species can be
clearly seen due to the differences in their electrophoretic mobilities.

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Fig. 5.
Metal affinity of p53tet complexes.
Combinations of His6-tagged and untagged p53tet
complexes captured using metal affinity resin (Talon) were analyzed by
SDS-PAGE. For each pair of p53tet constructs tested, the first lane
represents the mixture of both components prior to the addition of
Talon resin; the second lane is an aliquot of the supernatant after
incubation with Talon resin; and the third lane depicts the eluate
recovered in the presence of imidazole. a: lanes
1-3, His6-p53tet-E343K/E346K and
His6-p53tet-K351E; lanes 4-6,
p53tet-E343K/E346K and p53tet-K351E; lanes 7-9,
His6-p53tet-E343K/E346K and p53tet-K351E; lanes
10-12, p53tet-E343K/E346K and His6-p53tet-K351E.
b: lanes 1 and 3,
His6-p53tet-E343K and p53tet-K351E; lanes 4-6,
His6-p53tet-E346K and p53tet-K351E; lanes 7-9,
His6-p53tet-WT and p53tet-K351E; lanes 10-12,
His6-p53tet-WT and p53tet-E343K/E346K.
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The temperature melting curves for various p53tet combinations (Fig. 4)
indicate that the formation of a heterotetramer with p53tet-K351E was
most specific and strongest for the E343K/E346K double mutant. These
findings are also supported by the results from metal affinity capture
experiments. As Fig. 5b shows, His6-p53tet-E343K and His6-p53tet-E346K both interacted with p53tet-K351E,
resulting in the retention of p53tet-K351E on the Talon matrix
(lanes 1-3 and 4-6, respectively). However,
both experiments yielded a significant amount of cleaved p53tet-K351E
in the wash fractions (lanes 2 and 5). This was
almost as much as when a mixture of His6-p53tet-WT and
cleaved p53tet-K351E was evaluated (lanes 7-9). More
importantly, lanes 10-12 show that
His6-p53tet-WT did not bind to cleaved p53tet-E343K/E346K either, as a substantial amount of this protein was also found in the
unbound fraction. Thus, p53tet-E343K/E346K and p53tet-K351E form
heterotetramers with each other, but not with p53tet-WT.
 |
DISCUSSION |
The tetramerization domain of human p53 is an important part of
this key tumor suppressor protein. Analysis of the dimer-dimer interface of the human p53 tetramerization domain suggests that ion
pair interactions between Glu343, Glu346, and
Lys351 may contribute significantly to the stability of the
tetramer. This hypothesis was further supported by the fact that the
tetramerization domain sequences of p53 in other organisms as well as
of human p63 and p73 (Fig. 1c) display the naturally
occurring E343K mutation. This mutation is always coupled with a
corresponding loss of the positively charged lysine residue at position
351. This hypothesis was tested by designing and analyzing variants of
the tetramerization domain of human p53, viz. p53tet-E343K,
p53tet-E346K, p53tet-E343K/E346K, and p53tet-K351E, harboring
charge-reversal mutations at ionic residues.
In the first part of this study, the oligomeric state of these p53tet
mutants was evaluated (Fig. 3 and Table I) to determine whether these
mutations, as is the case with many mutations of hydrophobic residues
in the dimer-dimer interface (10, 12, 15), change the oligomerization
specificity of p53tet from a tetramer to a dimer. Indeed, it was
revealed that p53tet-K351E is a dimer in solution, demonstrating that a
single mutation to a charged residue is sufficient to produce dimeric
p53. This finding confirms our hypothesis that the introduction of a
charge-reversal mutation (Lys to Glu) at position 351 of the p53tet
domain introduces non-constructive charge repulsions at the dimer-dimer interface.
In the next part of this study, the stability of the resulting p53tet
mutants was evaluated. It has been established that the oligomeric
state and folding pattern of the p53 tetramerization domain are tightly
linked features of this protein scaffold, with the monomeric form being
essentially unfolded (10, 13, 32). Thus, thermal unfolding patterns as
measured by CD represent an indicator of the tendency of the p53tet
domain to oligomerize. As expected for a tetrameric protein, the
Tm of p53tet is dependent on protein concentration
(10, 32). A p53 monomer concentration of 10 µM was thus
selected for p53tet-WT to be fully unfolded at 98 °C, allowing us to
compare Tm values between p53tet-WT and its
variants. The Tm of p53tet is also dependent on the
length of the protein or peptide used (10). However, the observed
Tm of 68 °C for the 72-amino acid-long p53tet-WT
construct used in this study is comparable to published values for
related p53 constructs analyzed under these conditions (9, 10, 32). The
thermal unfolding results shown in Fig. 4 revealed that p53tet-E346K
was less stable than p53tet-E343K, suggesting that Glu346
is involved in a more pronounced stabilizing interaction at the dimer-dimer interface. Glu346 (rather than
Glu343) may thus more strongly interact with
Lys351, in contrast with predictions arising from the
crystal structure (6). CD thermal unfolding studies showed that
p53tet-K351E, in addition to being a dimer, was also very unstable,
suggesting that the charge at position 351 is an important determinant
of the stability of the p53 tetramer.
Subsequent experiments were undertaken to determine the potential of
these p53tet mutants to form heterotetramers. SEC (Fig. 3) and
analytical ultracentrifugation (Table I) data both indicated that
either the E343K or E346K mutation was sufficient to produce a species
that specifically formed heterotetramers with p53tet-K351E. However, CD
data (Fig. 4 and Table II) suggested that when both E343K and E346K
mutations were included, the resulting heterotetramer with p53tet-K351E
was much more stable relative to the two individual components. The
specificity of the heterotetramer between p53tet-E343K/E346K and
p53tet-K351E was also confirmed by metal affinity experiments (Fig. 5),
which strikingly depicted the necessity of both Glu-to-Lys mutations in
determining the specificity of the heterotetramer. It was also found
that these two mutants specifically associated with each other, and not
with wild-type human p53tet. This interesting finding suggests that
human p53 mutants containing such mutations would indeed not have a
`dominant-negative` effect on cellular transformation because their
intracellular expression would not directly compete or exchange with
existing cellular pools of wild-type human p53 (33, 34). This study
demonstrates for the first time the important contribution of ionic
interactions involving Glu343, Glu346, and
Lys351 to the stability of the dimer-dimer interface of the
human p53tet domain.