From the The Wistar Institute and the
¶ Department of Chemistry, University of Pennsylvania and the
§ Department of Biochemistry and Biophysics, University of
Pennsylvania School of
Medicine, Philidelphia, Pennsylvania 19104
Received for publication, December 22, 2000
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ABSTRACT |
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The p53 tumor suppressor is a sequence-specific
DNA-binding protein that activates transcription in response to DNA
damage to promote cell cycle arrest or apoptosis. The p53 protein
functions in a tetrameric form in vivo and contains four
domains including an N-terminal transcriptional activation domain, a
C-terminal regulatory domain, a tetramerization domain, and a central
core DNA-binding domain that is the site of the majority of
tumor-derived mutations. Here we report the 2.7-Å crystal structure of
the mouse p53 core domain. Like the human p53 core domain in complex
with DNA, the mouse p53 core domain adopts an immunoglobulin-like The p53 tumor suppressor protein functions as a checkpoint during
the G1/S cell cycle transition, responding to DNA damage by
activating transcription of genes that encode proteins inducing cell
cycle arrest or apoptosis (1, 2). Disruption of the G1/S
cell cycle transition and mutation of the p53 protein in particular
occur in a variety of cancer types and correlate with the majority of
human cancers (3).1
The majority of p53 tumor-derived mutations are now known to inactivate
its DNA binding properties and therefore to impair its ability to
activate transcription (4).
The transcriptional activity of p53 is mediated by a tetrameric
form of the protein that binds DNA in a sequence-specific fashion to
activate the transcription of target genes (5-8). Each p53 subunit
contains four functionally distinct domains: a loosely folded
N-terminal transcriptional activation domain (residues 1-44), a
central core (residues 102-292) containing a DNA-binding domain, a
tetramerization region (residues 320-356), and a regulatory domain
(residues 356-393) (4, 9, 10). There are over 100 naturally occurring
p53 DNA target sequences, and the human genome has been estimated to
contain about 200-300 of such sites (11). Although the sequences of
these p53 DNA target sites show variability, they all contain two head
to tail decamers each containing a pentameric inverted repeat. Most
decamers contain the consensus sequence
PuPuPuC(A/t) Significant insights into the tetramerization and DNA binding
properties of p53 are revealed by the structures of the isolated tetramerization domain (14-16) and of the human p53 core domain bound
as a monomer to DNA (4), respectively. The structure of the
tetramerization domain reveals a dimer of dimers. The structure of the
p53 core domain bound to DNA reveals the details of monomer binding to
a pentameric DNA sequence. These findings were used to rationalize the
functional consequence of the majority of tumor-derived p53 mutations
by showing that they map to regions of the core domain that would
either affect the stability of the core domain itself or destabilize
protein-DNA contacts. Despite the insights that are gained from the
structural analysis of p53, several questions underlying p53
structure/function still remain. Among these questions is the issue of
the nature of the structural rearrangements that p53 undergoes upon DNA binding.
We report here the crystal structure of the mouse p53 core domain in
the absence of DNA. A comparison with the crystal structure of the
human p53 core domain bound as a monomer to DNA reveals that the
overall structure of the core domain remains largely unperturbed upon
DNA binding except for a pronounced movement of a loop region. This
loop region adopts a conformation that is incompatible with DNA binding
in the DNA-free structure but adopts a conformation that facilitates
major groove DNA contacts in the DNA-bound structure. Moreover, the
crystals analyzed here, reveal a noncrystallographic trimer with three
nearly identical subunit-subunit (dimer) contacts. These dimer contacts
align the p53 core domains in a way that is incompatible with
simultaneous DNA binding by both protomers of the dimer. Interestingly,
similar dimer contacts are observed in crystals of the human p53 core domain with DNA in which only one of three p53 protomers in the asymmetric unit cell is specifically bound to DNA. We discuss the
implications of these findings for DNA-induced structural rearrangements of the p53 core domain in the context of the intact p53 tetramer.
Purification and Crystallization of the Mouse p53 Core
Domain--
The DNA sequence encoding the mouse p53 core domain
(residues 92-292) was amplified from the plasmid p11-4 (obtained from A. Levine, Princeton University) by polymerase chain reaction and subcloned into the pRSET (Invitrogen) bacterial expression vector.
The plasmid was transformed into the Escherichia coli BL21/DE-3 strain. Cells were initially grown in LB medium at
37 °C. When cultures reached an absorbance at 595 nm of
~0.4-0.6, cells were induced by the addition of 0.5 mM isopropyl-1-thio-
Crystallization attempts were initially directed at obtaining a complex
of the p53 core domain bound as a tetramer to a 24-base-paired DNA
duplex containing four pentameric DNA binding sites. To this end, 10 mg/ml protein was mixed with a 0.25 molar equivalent of DNA duplex and
screened for crystallization against a crystallization screen for
protein-DNA complexes using the hanging drop method. The best crystals
were obtained against a reservoir containing 8% polyethylene glycol
4000, 200 mM KCl, 50 mM
MgCl2, 10 mM dithiothreitol, and 50 mM Tris-HCl, pH 7.5, and grew to a typical size of 0.2 × 0.4 × 0.4 mm. Subsequent washing of large well formed crystals followed by crystal dissolution and analysis on SDS-polyacrylamide gel
electrophoresis and silver staining revealed that these crystals contained the p53 core domain but no DNA.
Data Collection, Structure Determination, and
Refinement--
Crystals of the mouse p53 core domain were
flash-frozen in a cryoprotectant containing the reservoir solution
supplemented with 25% polyethylene glycol 400, and diffraction
data were collected at 120 K at the A1 station of Cornell High Energy
Synchrotron Source using a charged coupled device detector. Data
were processed and scaled with MOLSFLM (17). Crystals belong to the
orthorhombic space group C2221 with three
molecules in the asymmetric unit. The structure was solved by molecular
replacement with the program AMoRe (18) using diffraction data from 10 to 3.0 Å. The search model used in the calculation was molecule B of
the human p53 core domain (accession number 1TSR). For the
search model, residues that differed between the human and mouse p53
core domains were changed to alanine (except for residues 183 and
prolines that were changed to glycines). Rotation and translation
searches followed by rigid body refinement yielded an unambiguous
solution for the three protomers in the asymmetric unit cell with an
R-factor of 47.5% and a correlation value of 42.4%.
The model was manually rebuilt against 2Fo Sedimentation Equilibrium Ultracentrifugation--
For
sedimentation equilibrium experiments, each cell was assembled with a
double sector 12-mm centerpiece with sapphire windows. Blank scans with
distilled water were taken before interference optics sedimentation
equilibrium experiments at appropriate speeds to correct for window
distortion of the fringe displacement data (24). Cells were loaded with
the mouse p53 core at two different starting concentrations (0.5 and
1.5 mg/ml) in a buffer containing 100 mM NaCl, 10 mM dithiothreitol, and 20 mM Tris-HCl, pH 7.5. The experiment was performed at three separate centrifugation speeds of
22,900, 32,400, and 37,500 rpm. At each speed, fringe displacement
scans were collected every 4 h until the protein samples reached
equilibrium. Equilibrium was assessed by comparison of successive scans
using the MATCH program, and data editing was performed using the
REEDIT program (both programs were provided by National Analytical
Ultracentrifugation Facility, Storrs, CT).
After equilibrium was obtained, the NONLIN program (25) was used
to globally fit the final scans from the two different concentrations and all speeds (a total of six curves were analyzed simultaneously). NONLIN fits used an effective reduced molecular weight, Overall Structure of the p53 Core Domain--
The crystal
structure presented here contains three molecules of the mouse p53 core
domain in the asymmetric unit cell. The overall fold of the p53 core
domain is very similar to that of the previously reported core domain
in complex with DNA (4) (Figs.
1A and 2A).
Briefly, the core domain forms a central region that adopts an
immunoglobulin-like
A superposition of the three molecules in the asymmetric unit (Fig.
2B) gives root mean square deviation values of 0.40 Å (molecule A-molecule B), 0.57 Å (molecule C-molecule A), and 0.62 Å (molecule B-molecule C) for all main chain atoms. Structural deviations
are largely restricted to the L1 loop (residues 115-121) along the
DNA-binding side of the core domain and the loops between strands S3 and S4 (residues 220-223) and S7 and S8 (residues 149-151) opposite to the DNA-binding side of the molecule. Although the structural variability within the S3-S4 and S7-S8 loops appears to
reflect inherent flexibility within this region of the core domain, as
will be discussed below, the structural variability within the L1 loop
appears to be due in part to the absence of bound DNA.
Comparison with the Human p53 Core Domain in Complex with
DNA--
The core domains of human and mouse p53 are highly homologous
in sequence with an overall identity of 89%. Therefore a comparison between the nascent mouse p53 core domain and the DNA-bound human p53
core domain can be used to examine the effect of core domain structure
as a function of DNA binding. An overall comparison of the nascent
(using monomer A) and the DNA-bound form of p53 shows a root mean
square deviation between main chain atoms of 0.87 Å as compared with a
value between 0.4 and 0.62 Å between noncrystallographically related
subunits of the nascent structure (Fig.
3A). The larger structural
differences that are observed when comparing the unbound and DNA-bound
forms of p53 within two different crystal forms are consistent with a
comparison between the p53 core domain in the absence of DNA with the
DNA-free p53 subunit within the crystal lattice of the p53-DNA complex
(Fig. 3C). This comparison shows a root mean square
deviation between main chain atoms of 0.57 Å. Taken together, DNA
binding by the p53 core domain appears to illicit small yet significant
structural changes.
The structural changes between the DNA-bound and DNA-free core domains
are primarily localized to four regions (Fig. 3A). Near the
DNA-binding surface structural differences are seen in the L1 loop and
the C-terminal end of the H2 helix. Away from the DNA-binding surface
structural differences are seen in the loop separating the S7 and S8
strands and the loop between the H1 helix and the S5 strand. Structural
differences within the S7-S8 and H1-S5 loops appear to be a function of
interactions between the p53 core domains in the crystals, as will be
discussed below, whereas structural differences within the L1 loop
appear to be a function of DNA binding.
The L1 loop in the DNA-free form of the p53 core is further away from
the H2 helix than it is in the DNA-bound form. In fact, a superposition
of the DNA-free p53 core onto the core domain of the DNA complex
suggests that the L1 loop would clash with the DNA and therefore
suggests that the L1 loop would need to move closer to the H2 helix (as
it is in the DNA-bound structure) to fit into the major groove of the
DNA. Two interactions within the L1 loop appear to stabilize its
DNA-bound form over the DNA-free form. Within the L1 loop, Lys-117
(Lys-120 in human p53) is disordered in all three core domains of the
asymmetric unit cell (Fig. 3B). In contrast, the DNA complex
shows that Lys-120 makes contacts to the major groove of the DNA. The
conformation of the L1 loop in the DNA bound form is also stabilized by
a backbone H bond at the turn of the L1 loop between Ala-116 and
Ser-118 (mouse p53 numbering), an interaction that is lost in the
DNA-free form (Fig. 3B). Taken together, the L1 loop region
of the p53 core appears to undergo important structural rearrangement
for DNA binding.
Dimer Contacts between p53 Core Domains in the Crystals--
The
most surprising finding from our crystal structure is that the mouse
p53 core domain is packed in the crystals as a noncrystallographic trimer in which the subunits are held together by nearly identical dimer contacts. These dimer contacts are quite extensive and bury a
total of ~1458 Å2 at each interface, which amounts to
~15% of the surface area of each protein subunit (Fig.
4A). These dimer contacts
involve the H1-S5 loop of one subunit and the S4-H1 (L2) and S6-S7
loops of the other dimer subunit (Fig. 4B). The interface is
stabilized by both hydrogen bonds and Van der Waals interactions. Main
chain hydrogen bonds are formed between residues 168 and 169 of the S4-H1 loop with residues 180 and 178 of the H1-S5 loop of the opposing
subunit, respectively, and between residue 207 of the S6-S7 loop with
residue 182 of the H1-S5 loop. Side chain hydrogen bonds are also
formed in the dimer, and Arg-178 of the H1-S5 loop plays a particularly
important role in this regard. The side chain of Arg-178 makes a direct
hydrogen bond to the backbone OH of residue 172 of the S4-H1 loop of
the opposing subunit and also makes water-mediated interactions to the
side chains of Glu-168 and Tyr-160 of the same loop. Van der Waals
interactions at the dimer interface involve interactions between
Phe-209 of the S6-S7 loop with Ala-182 and Pro-188 of the H1-S5 loop
and between Val-169 of the S4-H1 loop with the aliphatic regions of
Arg-178 and Ser-180 of the H1-S5 loop.
The dimer interaction in the crystals position the protein segments
involved in DNA interaction, the H2 helix and the L1 loop, in a
configuration that is incompatible with the simultaneous binding to
duplex DNA by both protomers of the dimer. Therefore the dimer in the
crystals represents a configuration that is inactive for DNA binding in
which each core domain binds a pentameric DNA sequence. The observation
that the noncrystallographic trimer mediates identical dimer contacts
indicates that the crystallographically observed dimer interface
represents a stable minimum. Interestingly, very similar dimer contacts
are observed in crystals of the human p53 core domain in the presence
of DNA. In these crystals, an asymmetric unit cell contains three p53
core domains and one DNA duplex. Although one p53 protomer is bound
specifically to a consensus pentameric DNA site, a second p53 protomer
is bound nonspecifically at the junction of two DNA duplexes, and a
third p53 protomer is not associated with DNA but is involved in dimer
contacts with the DNA-binding p53 protomer in the asymmetric unit cell.
Strikingly, these dimer contacts are very similar to the dimer contacts
that are observed in the DNA-free mouse p53 core domain crystals
reported here. Specifically, the same secondary structural elements are involved, the H1-S5 loop of one subunit and the S4-H1 (L2) and S6-S7
loops of the other subunit. Moreover, although the details of the
contacts are different, many of the same residues are used to stabilize
the dimer. Notably, Arg-174, Arg-181, and Glu-180 (analogous to
Arg-171, Arg-178, and Glu-177 in the DNA-free dimer) play important
roles in mediating H bonds within the dimer. Phe-212 and Pro-191
(analogous to Phe-209 and Pro-188 in the DNA-free dimer) also mediate
Van der Waals interactions within the dimer (Fig. 4D).
In addition to the common interactions within the dimer interface of
the DNA-free and DNA-containing p53 core domain crystals, there are
several divergent interactions resulting in a somewhat different
disposition of the two subunits of the dimer when the DNA-bound and
DNA-free forms are compared. The overall root mean square deviation
between the two p53 dimers is 5.5 Å (Fig. 4C). However, a
superposition of one of the two subunits reveals that the other
subunits of the corresponding dimers are related by a 12 ° rotation
of one relative to the other. Taken together, the dimer contacts
observed in two different p53 crystal lattices show striking
similarity, although there appears to be some flexibility in the
details of the interactions that stabilize the dimer.
Biological Implications of Dimer Contacts within the p53 Core
Domain--
In light of the similar p53 dimer contacts that are
present within the crystal lattices of the mouse and human p53 core
domain, we investigated the aggregation properties of the mouse p53
core domain in solution. To do this we carried out sedimentation
equilibrium ultracentrifugation using two different protein
concentrations and three different centrifugation speeds. Analysis of
the data revealed a sedimentation profile that could be best fit by a
monomer-dimer model fixing the molecular mass to the size of
22,695 Da with a Kd of 2.1 mM for low
levels of dimer formation (Fig. 5). This
model for slight oligerimerization, characterizing reversible monomer-dimer equilibrium, was significantly better than a single species model or models describing monomer-trimer or
monomer-dimer-tetramer equilibrium. Taken together, the
sedimentation equilibrium ultracentrifugation results clearly
demonstrate that the isolated mouse p53 core domain is predominantly
monomeric at physiological protein concentrations.
In light of these results, what can be the biological implications of
the p53 dimer contacts that are observed in the crystals? In
vivo p53 exists in a tetrameric form largely due to the presence of a highly conserved C-terminal tetramerization domain. Therefore the
four in vivo p53 core domains are at high local
concentrations and furthermore have an enhanced propensity to form
specific interactions with each other. Given the tetrameric form of the
p53 protein, the most likely interactions between the core domains
would be either a dimer of dimers (as observed for the isolated
tetramerization domain) or a symmetrical tetramer. For
sequence-specific DNA binding, p53 has also been proposed to undergo a
conformational change from a state with low affinity for DNA (T state)
to a state with high affinity for DNA (R state) (6, 13). These
different physiological states of p53 have been proposed based on the
activities of antibodies that specifically react with and enhance one
of the two states. Based on p53 DNA target sites that are two head to
tail decamer repeats of 2-fold symmetric pentameric sites, the p53
conformation with high affinity for DNA is proposed to have the core
domains aligned as two 2-fold symmetric dimers. Based on our
crystallographic results, we propose that in the context of the p53
tetramer the core domain is arranged as a dimer of dimers and that the
core domain dimer observed in our crystals represents a low affinity
DNA state. This is consistent with the observation that the dimer in
our crystals is configured in such a way that it is incompatible with
simultaneous binding to duplex DNA. It is also consistent with the
observation that a similar dimer is observed in the crystals of the
human p53 core domain in the presence of DNA and in which only one of
two subunits of the dimer make specific contacts to DNA. An
electrostatic potential energy surface of the p53 core domain dimer in
our crystals revealed a pronounced electropositive charge patch along
one face of the dimer (Fig. 6). This
surface may form a docking site for an acidic region of the p53 protein
such as the N-terminal transactivation domain in the low affinity DNA
binding state. Such an interaction would also serve to mask the
transactivation domain of p53 prior to high affinity DNA binding.
In conclusion, we have determined the crystal structure of the mouse
p53 core domain and have compared it with the DNA-bound form of its
human homologue. The comparison reveals that DNA binding by the core
domain is accompanied by the reconfiguration of a loop region for major
groove interactions with the DNA. Strikingly, we observe dimers of p53
core domains in our crystals in a configuration that is incompatible
with simultaneous binding of both subunits to duplex DNA. A comparison
with a similarly configured dimer in crystals of the human p53 core
domain suggests that this dimer may represent a physiologically
relevant low affinity DNA binding state of the p53 core domains in the
context of the intact p53 tetramer. Further insights into the
structural rearrangements mediated by p53 as a function of DNA binding
will require structural analysis of the intact p53 tetramer in the
presence and absence of DNA.
sandwich architecture with a series of loops and short helices at
opposite ends of the
sandwich. Comparison of the DNA-bound and
DNA-free p53 core domains reveals that while the central
sandwich
architecture remains largely unchanged, a loop region important for DNA
binding undergoes significant rearrangement. Although this loop region
mediates major groove DNA contacts in the DNA-bound structure, it
adopts a conformation that is incompatible with DNA binding in the
DNA-free structure. Interestingly, crystals of the DNA-free core domain
contain a noncrystallographic trimer with three nearly identical
subunit-subunit (dimer) contacts. These dimer contacts align the p53
core domains in a way that is incompatible with simultaneous DNA
binding by both protomers of the dimer. Surprisingly, similar dimer
contacts are observed in crystals of the human p53 core domain with DNA
in which only one of the three p53 protomers in the asymmetric unit
cell is specifically bound to DNA. We propose that the p53 core domain dimer that is seen in the crystals described here represents a physiologically relevant inactive form of p53 that must undergo structural rearrangement for sequence-specific DNA binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(T/a)GPyPyPy, where Pu and Py are purines and
pyrimidines, respectively (12). Although the various domains of the p53
protein can function autonomously, in vivo activity requires
the intact protein. In addition, various lines of evidence suggest that
the intact protein exists in two conformational states, one that has
low affinity for DNA (T state) and another that has a high affinity for
DNA (R state) (6, 13).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-D-galactopyranoside (supplemented with 100 µM zinc acetate) and grown
overnight at 15 °C. The cells were isolated by centrifugation and
resuspended in low salt buffer A (20 mM sodium citrate, pH
6.1, 100 mM NaCl, 10 µM Zn2+, and
10 mM dithiothreitol). The cells were sonicated, and
cellular debris was removed by centrifugation. The supernatant
containing soluble mouse p53 core domain was loaded onto a SP-Sepharose
(Amersham Pharmacia Biotech) ion exchange column, which was washed with 20× column volumes of buffer A, and the protein was eluted with a
0.1-1.0 M NaCl gradient. Peak fractions containing p53
core domain were pooled and concentrated using a Centriprep-10
(Amicon) and further purified by gel filtration chromatography using a Superdex-75 column. Peak fractions were judged to be greater than 97%
homogeneous by SDS-polyacrylamide gel electrophoresis analysis, concentrated to ~40-55 mg/ml, and stored at
80 °C until further use.
Fc, Fo
Fc,
and Fc
Fo maps using the
program O (19). Rigid body refinement, least square minimization, and
simulated annealing refinement with crystallography and NMR
suite (20) using noncrystallographic symmetry restraints between
the three protomers in the asymmetric unit cell were carried out at a
resolution range of 10-3.0 Å. When the data were extended to a 2.7-Å
resolution, noncrystallographic symmetry restraints were gradually
released, and several cycles of simulated annealing (21), torsion angle
dynamic (22), temperature factor refinement, and manual model building
were carried out. During the final stages of refinement, a bulk solvent
correction was applied to the data (23), water molecules were included
using the waterpick routine of crystallography and NMR suite,
and three zinc ions were added. Some residues at the very N and C
termini were not visible in the electron density map and were therefore
not modeled. The final structure contained residues 99-284 of each
protomer, had excellent stereochemistry, and had crystallographic
R-factors of 23.9 and 29.9% for R-working and
R-free, respectively, against data from 10 to 2.7 Å (see
Table I and Fig. 1B).
Data collection and refinement statistics
= M(1
)
2/RT, in which
M is the molecular weight,
is the solvent density,
is the angular
velocity (2
(rpm)/60), R is the gas constant, and
T is the temperature in Kelvin. The partial specific volume
of the mouse p53 core was calculated from the amino acid sequence, and
the density of the solvent was calculated as described previously (26).
For this model of weak self-association,
was held at the correct
value based on the known monomer molecular weight of the protein, and
separate equilibrium constants for each scan were fitted as ln K. These
values were converted to dissociation constants with the appropriate
molar units. The fit quality for the model was determined by
examination of residuals and by minimization of the fit variance. The
root mean square deviation of the residuals for the multiple species
model was 9.56 × 10
3.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
sandwich architecture of two long twisted
antiparallel
sheets of four (S1, S3, S8, and S5) and five (S10, S9,
S4, S7, and S6) strands (Figs. 1A and
2A). Located at opposite ends
of the
sandwich are a series of loops. One of the two ends also
contains two short helices (H1 and H2) and a tightly bound zinc atom
that is tetrahedrally ligated by Cys-173 and His-176 from the L2 loop
(between strands S4 and S5) and Cys-235 and Cys-239 from the L3 loop
(between strands S8 and S9). The structure of the human p53 core domain
bound to DNA shows that the H2 helix, the L3 loop, and the L1 loop
(between strands S1 and S2) interact with DNA.
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Fig. 1.
A, sequence alignment of the p53
core domains from mouse and human. Sequences are aligned using the
CLUSTAL program and displayed with the BOXSHADE program.
Black and gray backgrounds are used to indicate
identical and conserved residues at a given position, respectively.
Secondary structural elements within the mouse p53 core domain are
shown above the sequence alignment. The circle symbols
indicate residues that are associated with the dimer interface of the
mouse DNA-free (black circle) and human DNA-bound
(gray circle) p53 core domain, and the * symbol indicates
residues of the human p53 core domain that contact DNA in the human p53
core-DNA complex. B, representative Fo Fc omit electron density map of the refined mouse
p53 core domain structure proximal to the dimer interface. The map is
contoured at 2.5
. The green and red colors
indicate molecules A and B of the p53 core domain within the asymmetric
unit cell.
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Fig. 2.
Overall structure of the mouse p53 core
domain. A, schematic ribbon diagram of the p53 core
domain. B, C superposition of the three p53 protomers in
the asymmetric unit cell of the crystals.
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Fig. 3.
Comparison of the DNA-free form of the mouse
p53 core domain with the DNA-bound and DNA-free forms of the human p53
core domain. The DNA-free mouse p53 core domain is shown in
green, and the human p53 core domain is shown in
red. A, C superimposition of the DNA-free
mouse p53 core domain with the DNA-bound form of the human p53 core
domain. The DNA that is bound to the human p53 core domain is shown in
blue. B, close-up view of the p53-DNA interface
proximal to the L1 loop (A). The DNA-bound form of the human
p53 core domain is shown in red, highlighting residues
116-119 of the L1 loop (mouse p53 numbering). The corresponding region
of molecule A of the DNA-free mouse p53 core domain is shown in
gray. The conformations of the Lys-117 side chain in the
three mouse p53 core domains in the asymmetric unit cell are shown in
dark gray. C, C
superimposition of the
DNA-free mouse p53 core domain with the DNA-free form of the human p53
core domain.
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Fig. 4.
Structure of the mouse p53 core domain
dimer. A, trimeric packing of the mouse p53 core domain
in the asymmetric unit cell viewed perpendicular to the 3-fold axis of
the trimer. B, stereo diagram showing the intermolecular
interaction at the dimer interface of the trimeric structure. Protomer
A is shown in green, and protomer B is shown in
blue. C, superposition of the mouse p53 core
domain dimer (using protomers A and B) with the p53 core domain dimer
in crystals of the human core domain in which only one of the two
protomers is specifically bound to DNA. The left view shows
an overall superposition of the dimers, and the right view
shows a superposition of only one of the protomers of the dimer.
This type of superposition shows that the second protomers of the
respective dimers are related by a 12 ° rotation. D,
dimer contacts within the crystals of the human p53 core domain in
which only one of the two protomers is specifically bound to DNA.
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Fig. 5.
Equilibrium sedimentation ultracentrifugation
of the mouse p53 core domain. Two different initial protein
concentrations (0.5 mg/ml (lower curve) and 1.5 mg/ml
(upper curve)) were run at each of three different
centrifugation speeds (as indicated in the headings). The
bottom panels show concentration distribution plots along
the filtered curves assuming a single species.
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Fig. 6.
Electrostatic surface of the mouse p53 core
domain dimer. The electrostatic potential is calculated with the
program GRASP (Nicholls et al., 1991) and displayed
as a color gradient from red (electronegative
10
kilotesla/charge) to blue (electropositive,
10
kilotesla/charge). A and B indicate subunits A
and B of a noncrystallographic dimer of the unit cell.
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ACKNOWLEDGEMENT |
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We thank Irina Kriksunov and Marian Szebenyi for help on beamline A1 at Cornell High Energy Synchrotron Source and R. Burnett for useful discussions.
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FOOTNOTES |
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* This work was supported by Grant DAMD17-99-1-9456 from the United States Army Breast Cancer Research Program (to R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1HU8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: The Wistar Inst.,
3601 Spruce St., Philadelphia, PA 19104. Tel: 215-898-5006; Fax:
215-898-0381; E-mail: marmor@wistar.upenn.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M011644200
1 Contact the corresponding author for an additional Web address.
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