The dihedral symmetry of the p53 tetramerization domain mandates a conformational switch upon DNA binding.

Abstract

The p53 tumor suppressor forms stable tetramers, whose DNA binding activity is allosterically regulated. The tetramerization domain is contained within the C-terminus (residues 323-355) and its three-dimensional structure exhibits dihedral symmetry, such that a p53 tetramer can be considered a dimer of dimers. Under conditions where monomeric p53 fails to bind DNA, we studied the effects of p53 C-terminal mutations on DNA binding. Residues 322-355 were sufficient to drive DNA binding of p53 as a tetramer. Within this region residues predicted by the three-dimensional structure to stabilize tetramerization, such as Arg337 and Phe341, were critical for DNA binding. Furthermore, substitution of Leu344 caused p53 to dissociate into DNA binding-competent dimers, consistent with the location of this residue at the dimer-dimer interface. The p53 DNA site contains two inverted repeats juxtaposed to a second pair of inverted repeats. Thus, the four repeats exhibit cyclic-translation symmetry and cannot be recognized simultaneously by four dihedrally symmetric p53 DNA binding domains. The discrepancy may be resolved by flexible linkers between the p53 DNA binding and tetramerization domains. When these linkers were deleted p53 exhibited novel DNA binding properties consistent with an inability to recognize four contiguous DNA repeats. Allosteric regulation of p53 DNA binding may involve repositioning the DNA binding domains from a dihedrally symmetric state to a DNA-bound asymmetric state.

Full text

Page 1

The EMBO Journal vol.14 no.3 pp.512-519, 1995
The dihedral symmetry of the p53 tetramerization
domain mandates a conformational switch upon
DNA binding
Jennifer L.F.Waterman, Jill L.Shenk and
Thanos D.Halazonetis1
Department of Molecular Oncology, The Wistar Institute, Philadelphia,
PA 19104-4268
'Corresponding author
J.L.F.Waterman and J.L.Shenk contributed equally to this work
Communicated by P.A.Sharp
The p53 tumor suppressor forms stable tetramers,
whose DNA binding activity is allosterically regulated.
The tetramerization domain is contained within the C-
terminus (residues 323-355) and its three-dimensional
structure exhibits dihedral symmetry, such that a p53
tetramer can be considered a dimer of dimers. Under
conditions where monomeric p53 fails to bind DNA,
we studied the effects of p53 C-terminal mutations on
DNA binding. Residues 322-355 were sufficient to drive
DNA binding of p53 as a tetramer. Within this region
residues predicted by the three-dimensional structure
to
stabilize tetramerization, such as Arg337 and
Phe341, were critical for DNA binding. Furthermore,
substitution of Leu344 caused p53 to dissociate into
DNA binding-competent dimers, consistent with the
location of this residue at the dimer-dimer interface.
The p53 DNA site contains two inverted repeats juxta-
posed to a second pair of inverted repeats. Thus, the
four repeats exhibit cyclic-translation symmetry and
cannot be recognized simultaneously by four dihedrally
symmetric p53DNA binding domains. The discrepancy
may be resolved by flexible linkers between the p53
DNA binding and tetramerization domains. When
these linkers were deleted p53 exhibited novel DNA
binding properties consistent with an inability to recog-
nize four contiguous DNA repeats. Aliosteric regulation
of p53 DNA binding may involve repositioning the
DNA binding domains from a dihedrally symmetric
state to a DNA-bound asymmetric state.
Key words: conformational shift/DNA binding/oligomeriza-
tion/p53/tumor suppressor
Introduction
Wild-type p53 is a sequence-specific DNA binding protein
inactivated
in more than half of all human tumors
(reviewed by Friend, 1994). p53 binds DNA as a tetramer
(Kraiss et al., 1988; Stenger et al., 1992; Sturzbecher
et al., 1992; Friedman et al., 1993; Halazonetis and Kandil,
1993; Hainaut et al., 1994). Its DNA binding domain has
been mapped to residues 90-290 (Bargonetti et al., 1993;
Halazonetis and Kandil, 1993; Pavletich et al., 1993;
Wang et al., 1993). The N-terminus encodes a transcription
activation domain (Fields and Jang, 1990; Unger et al.,
52Oxford University Press
512
1992; Lin et al., 1994), while the C-terminus mediates
oligomerization (Milner et al., 1991; Sturzbecher et al.,
1992; Iwabuchi et al., 1993; Pavletich et al., 1993) and
regulates allosterically the DNA binding activity of p53
(Hupp
et
1992; Halazonetis and Kandil,
Halazonetis et al., 1993). These two biochemical activities
of the p53 C-terminus map to distinct regions. Deletion
of residues 364-393 activates DNA binding (Hupp et al.,
1992),
but
has
no
effect
(Halazonetis and Kandil, 1993).
The C-terminus regulates DNA binding of p53 by
controlling the conformation of p53 tetramers between
two states: an R state with high affinity for DNA and a
T state with no or low affinity for DNA (Halazonetis and
Kandil, 1993; Halazonetis et al., 1993). In the absence of
DNA the equilibrium between the two states is shifted
towards the T state; DNA binding thus requires an
energetically unfavorable conformational switch to the R
state. Antibody PAb421, whose epitope maps to residues
373-381 of human p53, or deletion of residues 364-393
of human p53, shifts the conformation to the R state and
activates DNA binding (Funk et al., 1992; Hupp et al.,
1992; Halazonetis and Kandil, 1993; Halazonetis et al.,
1993).
In addition to the regulatory region described above,
the p53 C-terminus is significant forDNA binding because
it mediates
oligomerization.
systems p53 oligomerization is required for sequence-
specific DNA binding. For example, in vitro translated
p53 does not bind DNA as a monomer (Halazonetis and
Kandil, 1993; Hainaut et al., 1994). The significance of
oligomerization for DNA binding relates to the nature of
the p53 DNA site, which contains four pentanucleotide
repeats (Bargonetti et al., 1991; Kern et al., 1991; El-
Deiry et al., 1992; Funk et al., 1992; Halazonetis et al.,
1993). Since each p53 DNA binding domain recognizes
one repeat (Halazonetis and Kandil, 1993; Cho et al.,
1994), a polyvalent p53 tetramer has greater avidity for
the p53 DNA site than a p53 monomer. Consistent with
this interpretation, at high protein concentrations, which
cannot be achieved by in vitro translation, monomeric p53
can bind DNA (Bargonetti et al., 1993; Friedman et al.,
1993; Pavletich et al., 1993; Wang et al., 1993). Whether
tetramerization facilitates p53 DNA binding in vivo has
not been definitively established. Nevertheless, the physio-
logically relevant p53 DNA sites contain four pentanucleo-
tide repeats (Kastan et al., 1992; El-Deiry et al., 1993;
Wu et al., 1993).
Oligomerization is also critical for the ability of p53
mutant proteins to inactivate wild-type p53. Hetero-
oligomers formed between p53 mutants and wild-type p53
are unable to bind DNA, and thus unable to regulate
cellular
proliferation
(Milner
Bargonetti et al., 1992; Farmer et al., 1992; Kern et al.,
1992).
al.,
1993;
on p53
oligomerization
In
certain experimental
andMedcalf,
1991;

Page 2

p53 conformational switch
Very recently the minimal region required for p53
tetramerization was mapped to residues 322-355 (Wang
et al.,
1994; Sakamoto et al.,
dimensional structure determined by NMR spectroscopy
(Clore et al., 1994; C.Arrowsmith, personal communica-
tion). Each subunit
structure elements: a , strand, a tight turn and an a helix.
Two subunits form a dimer via antiparallel interaction of
their ,B strands and two dimers interact through hydro-
phobic and electrostatic contacts between their a helices
to form a tetramer. Thus, the tetramerization domain
exhibits dihedral symmetry.
In what follows we characterize the DNA binding
activities ofp53 proteins with mutant C-termini to examine
if their biochemical properties are consistent with the
reported three-dimensional structure. In addition we con-
sider the implications of the oligomerization domain
symmetry for p53 DNA binding, and suggest that the N-
terminal third of the p53 C-terminus (residues 290-321),
like the C-terminal third (residues 364-393), is involved
in allosteric regulation of DNA binding.
1994) and
its three-
is comprised of three secondary
Results
Functional mapping of the p53 tetramerization
domain
The tetramerization domain is contained within the C-
terminus of p53 (Milner et al., 1991; Sturzbecher et al.,
1992; Iwabuchi et al.,
Sakamoto et al., 1994; Wang et al., 1994). Since in vitro
translated p53 does not bind DNA
(Halazonetis and Kandil, 1993; Hainaut et al., 1994), the
tetramerization domain can be finely mapped by analysis
of p53 C-terminal mutants using DNA binding as a
functional readout for oligomerization. We tested binding
to oligonucleotide BC.V4A, which differs at one nucleo-
tide position from the optimal p53 DNA site (Funk et al.,
1992; Halazonetis et al., 1993). Wild-type p53 binds to
this site. DNA binding is further enhanced by antibody
PAb421, which shifts the conformation of p53 to the R
state (Funk et al., 1992; Hupp et al., 1992; Halazonetis
and Kandil, 1993; Halazonetis et al., 1993). Deletions of
residues 300-308, 300-317, 300-321, 379-393, 364-393
and 356-393 maintained and even enhanced sequence-
specific DNA binding, while deletions of residues 300-
327, 324-332 and 352-393 reduced binding to undetect-
able or barely detectable levels (Figure
implicating
residues
322-355
oligomerization-dependentDNA binding.
Dimeric forms of in vitro translated p53 can bind DNA
(Halazonetis and Kandil, 1993; Hainaut et al., 1994).
Thus, residues 322-355 might mediate formation ofdimers
rather than tetramers. We therefore determined the subunit
stoichiometry with which a deletion mutant lacking
residues 300-321 and 356-393 bound DNA. DNA com-
plexes of N-terminally truncated p53 migrate faster than
those with an intact N-terminus (Halazonetis and Kandil,
1993; Figure IB). Oligomerization stoichiometry can be
determined by examining the migrationofDNA complexes
of N-terminally intact and truncated p53 hetero-oligomers
(Hope and Struhl,
cotranslated p53A300-321A356-393 and p53A3-79A300-
321A356-393 were subjected to electrophoresis, three
1993; Pavletich et al., 1993;
as
a monomer
1A), thereby
necessary
as
for
1987). When DNA complexes of
A
PAb
42 1
Protein
None
p53wt
p53A379-393
p53A364-393
p53A356-393
p53A352-393
p53A324-332
p53A300-308
p53A300-31 7
p53A300-321
p53A300-327
B
+
+
+
+
+
+
0
_
9
_
Protein
None
p53 \300-321.N356-393
Cotranslation
Cotranslation
Cotranslation
p53x3-79A300-321A\356-393
3:1
1:1
1:3
Fig. 1. Functional mapping of the p53 tetramerization domain.
(A) In vitro translated p53 deletion mutants were assayed for binding
to 32P-labeled oligonucleotide BC.V4A by native gel electrophoresis.
Wild-type p53 and mutants retaining the epitope of antibody PAb421
were also tested for DNA binding in the presence of this antibody.
The names of the mutants indicate the deleted residues. (B) Subunit
stoichiometry conferred by residues 322-355. p53A300-321A356-393
and p53A3-79A300-321A356-393 were translated individually or
cotranslated at 3:1, 1:1 and 1:3 mRNA ratios. The translated proteins
were examined for DNA binding to 32P-labeled oligonucleotide BC.
To achieve adequate resolution, electrophoresis was continued until the
free 32P-labeled DNA had migrated off the gel.
novel bands were observed (Figure iB). Thus, residues
322-355, which form stable tetramers in vitro (Sakamoto
et al., 1994; Wang et al., 1994), are sufficient to mediate
DNA binding of p53 as a tetramer.
Defining critical structural determinants for p53
tetramerization
The p53 tetramerization domain contains three secondary
structure elements: a
326 and 334, characterized by alternating hydrophobic
and polar amino acids; a turn between residues 335 and
336 and an amphiphilicahelix extending between residues
337 and 355. A number of hydrophobic and electrostatic
interactions are predicted to stabilize this domain (Clore
et al., 1994; C.Arrowsmith, personal communication). To
evaluate them we examined DNA binding, which
oligomerization dependent, and oligomerization of tetra-
merization domain amino acid substitution mutants.
We initially focused on the tightturn spanningresidues
335-336 and its adjacent amino acids (Figure 2).A single
substitution of Gly334 by Gln and a double substitution
of Arg335 and Glu336 by glycines compromised,but did
not abolish DNA binding. A triple substitution ofGly334,
3strand extending between residues
is
513
I

Page 3

J.L.F.Waterman, J.L.Shenk and T.D.Halazonetis
PAb
42 1Protein
None
-
p53wt
+
0
p53G335-6
+
I
p53iG334
+
p53Q334
p53L337
A
PAb
42 1
Protein
None
p53wt
p53 13 2 7
p53A328
p53A338
p53A341
p53A341A344
p53L341
p53A351
p53A344
p53F334L337
p53L3341337
p53AQA334-6
+
+
+
+
+
Fig. 2. Effect of amino acid substitutions within the turn of the
tetramerization domain on DNA binding of full-length p53. In vitro
translated p53 was assayed for binding to 32P-labeled oligonucleotide
BC.V4A by native gel electrophoresis in the presence or absence of
antibody PAb421. The names of the mutants indicate the positions of
substitutions and the amino acids introduced using the single letter
amino acid code. Mutant p53iG334 has an insertion of a glycine at
position 334.
Arg335 and G1u336 with Ala, Gln and Ala, respectively,
abolished DNA binding. So did three mutations that
targeted Arg337: a single substitution of Arg337 by Leu,
and two double substitutions of Gly334 and Arg337
with Leu and Ile, respectively, and with Phe and Leu,
respectively. Insertion of a Gly between residues 334 and
335 also eliminated DNA binding (Figure 2).
Subsequently we targeted amino acids within the
strand and a helix (Figure 3A). Substitutions of Tyr327
or Phe328 within the ,3 strand by Ile and Ala, respectively,
did not compromise DNA binding. Substitutions of several
residues of the a helix also did not compromise DNA
binding. Thus, mutating Phe338 to Ala, Leu350 to Ala,
Lys351 to Ala or Leu, Gln354 to Arg, Ala355 to Leu,
and Gly356 to Ala did not significantly affect DNA
binding (Figure 3A and data not shown). In contrast,
substitutions of Phe341
binding to oligonucleotide BC.V4A (Figure 3A). Phe341
and Leu344 were subsequently targeted for further muta-
genesis. Substitution ofPhe341 by Leu did not compromise
DNA binding (Figure 3A). Neither did a double substitu-
tion of Phe341 and Leu344 by Leu and Ile, respectively
(Figure 3B). Substitution of Phe341 by Ile compromised
p53 DNA binding slightly, but consistently (Figure 3B).
We have previously described a mouse p53-GCN4
chimeric protein that substitutes the 47 C-terminal p53
residues, which include part of the p53 tetramerization
domain, with the leucine zipper of the GCN4 yeast
transcription factor (Halazonetis and Kandil, 1993). Oligo-
merization of this fusion protein is dependent on the
GCN4 leucine zipper, a well-characterized dimerization
domain (Hope and Struhl, 1987; Landschulz et al., 1988).
However,
thechimeric
(Halazonetis and Kandil, 1993). The equivalent human
p53 chimeric protein, p53LZ346, also exhibited sequence-
specific DNA binding characteristic of tetramers (see
Figure 5A below). If p53 residues drive p53LZ346 tetra-
,
or Leu344 by Ala abolished
protein
forms
tetramers
B
Protei n
None
p53wt
p53A341
p531341
p53L341 1344
p53LZ346
p53LZ346A341
p53LZ3461341
p53LZ346L341 1344
Fig. 3. Effect of amino acid substitutions within the
helix of the tetramerization domain on DNA binding of full-length
p53. In vitro translated p53 was assayed for binding to 32P-labeled
oligonucleotide BC.V4A by native gel electrophoresis in the presence
or absence of antibody PAb421. The names of the mutants indicate the
positions of substitutions and the amino acids introduced using the
single letter amino acid code. The mutations were introduced in the
context of wild-type p53 (A), or additionally in the context of
p53LZ346, which contains a GCN4 leucine zipper after residue 346 of
human p53 (B).
strand and a
merization, then mutations that affect wild-type p53 will
also affect p53LZ346. Indeed, substitutions of Phe341 by
Ala, Ile or Leu, affected similarly the DNA binding of
wild-type p53 and p53LZ346 (Figure 3B), suggesting that
both proteins have similar hydrophobic cores at their
tetramerization interfaces.
Oligomerization is required for DNA binding of in vitro
translated p53 (Halazonetis and Kandil, 1993; Hainaut
et al., 1994). We have therefore concluded that the ability
of the tetramerization domain mutants to bind DNA
reflects their ability to oligomerize. To strengthen this
conclusion, the ability of select mutants to oligomerize
was tested directly. We have demonstrated previously that
deletion of the 30 C-terminal amino acids of mouse p53
does not compromise tetramerization and furthermore that
this C-terminally truncated protein oligomerizes with full-
length p53 (Halazonetis and Kandil, 1993). These observa-
tions formed the basis for assaying the ability of human
p53 mutants to oligomerize. Human p53A364-393 was not
precipitated by antibody PAb421, whose epitope resides
within the deleted p53 C-terminus (Wade-Evans and
Jenkins,
1985;
Sturzbecher
et
al.,1992).
However,
514
S

Page 4

p53 conformational switch
Protein
Precip.
N4one
p53w t
p53 \364-393
p53wt+p53 \364-393
None
None
None
PAb421
None
PAb421
Control
None
p53F334L337+
p53F334L337x364-393 PAb421
p53A341+
p53A341.%364-393
p53wt+p53\352-393
None
PAb421
None
PAb421
I
I
S:
f
I
Fig. 4. Oligomerization of p53 proteins with deletions or amino acid
substitutions within the p53 C-terminus. 35S-labeled in vitro translated
proteins were translated individually or cotranslated as indicated.
Following translation the proteins were either immunoprecipitated with
antibody PAb42l, or with no antibody (Control) or not
immunoprecipitated (lanes marked None) and then analysed by SDS
gel electrophoresis.
p53A364-393 was precipitated when cotranslated with
wild-type p53, indicating that the two proteins oligomerize
(Figure 4). When full-length and C-terminally truncated
versions of mutant Phe334-Leu337 were cotranslated,
only the full-length version was precipitated by antibody
PAb421 (Figure 4). Substitution of Phe341 by Ala, or
deletion of residues 352-393 also abrogated coprecipita-
tion (Figure 4). Thus, for every tetramerization domain
mutant examined, oligomerization-dependent DNA bind-
ing accurately reflected their ability to oligomerize in the
absence of DNA.
Substitution of Leu344 causes p53 tetramers to
dissociate into dimers
A number of amino acid substitutions within the p53
oligomerization domain eliminated binding to oligonucleo-
tide BC.V4A, which contains a suboptimal p53 DNA site
(Figures 2 and 3A). These mutants were also tested for
binding to oligonucleotide BC, which contains the optimal
site (Halazonetis et al., 1993). They all failed to bind
(data not shown), with the exception of Ala344 (Figure
SA). Interestingly, the DNA complex of the latter mutant
migrated significantly faster than the DNA complexes of
wild-type p53 or p53A364-393 (Figure SA and data
not shown).
To determine the subunit stoichiometry of p53Ala344,
we studied its binding patterns to oligonucleotides con-
taining four pentanucleotide
arranged head-to-head or tail-to-tail (oligonucleotides BC,
BC.H1 and BC.H2, respectively). As controls we used
p53A364-393
and
two p53-GCN4
chimeric proteins: p53LZ346 and p53LZ335. The subunit
stoichiometries of the equivalent mouse p53 proteins have
been determined by analysis of their migration on native
polyacrylamide gels in the absence of DNA and by their
DNA binding specificities (Halazonetis and Kandil, 1993).
Consistent with the subunit stoichiometries previously
reported,
p53LZ346 were tetrameric, since they recognized the full-
repeats,
or two repeats
leucine
zipper
the human p53 proteins p53A364-393 and
A
Protein
32P DNA
None
BC
BC.H1
BC.H2
BC
BC.H1
BC.H2
BC
BC.H1
BC.H2
BC
BC.H1
BC.H2
BC
BC.H1
BC.H2
p53A364-393
p53A344
p53LZ346
p53LZ335
B
Protein
None
p53A344
Cotranslation
Cotranslation
Cotranslation
p53A3-79A 344
3:1
1:1
1:3
I
I
I
t
I
Fig. 5. Substitution of Leu344 by Ala causes p53 to dissociate into
DNA binding-competent dimers. (A) Binding of p53A364-393,
p53Ala344, p53LZ346 and p53LZ335 to 32P-labeled oligonucleotides
containing four (BC) or only two specific pentanucleotides (BC.H1
and BC.H2). (B) p53Ala344 and p53A3-79Ala344 translated
individually or cotranslated at 3:1, 1:1 and 1:3 mRNA ratios were
examined for binding to 32P-labeled oligonucleotide BC. To achieve
adequate resolution electrophoresis was continued until the free
32P-labeled DNA had migrated off the gel.
length DNA site with higher affinity than the half-length
sites, especially BC.H1, while p53LZ335 was dimeric,
since it bound the full-length and the BC.H1 half-length
sites with equal affinities, and failed to recognize the
BC.H2 site (Figure 5A). The binding pattern of dimeric
p53 reflects the unique orientation of the DNA binding
domains relative to one another which is compatible only
with pentanucleotides arranged head-to-head (Halazonetis
and Kandil, 1993). The DNA binding pattern ofp53Ala344
matched that of the dimeric p53LZ335 (Figure 5A).
As a second approach to determine the subunit stoichio-
metry of p53Ala344, we examined the DNA complexes
of p53Ala344 cotranslated with an N-terminally truncated
version of the same mutant (Hope and Struhl, 1987). The
full-length DNA site (oligonucleotide BC) was used to
avoid biasing the experiment. Three different ratios of
p53Ala344 and p53A3-79Ala344 mRNAs were used for
the cotranslations. At all mRNA ratios tested,cotranslation
yielded only one novel p53-DNA complex (Figure SB),
further supporting the conclusion that p53Ala344 is a
dimer.
515

Page 5

J.L.F.Waterman, J.L.Shenk and T.D.Halazonetis
Protein
None
\300-327
300-32 1
3-79\300-321
300*321 +3-79\300-321
.3-79 \300-327
300 327+.\3-79. 300 327
!290-297
\290-297\300- 3 21
None
|\300-321
X\300-3 27
.3-79\300-327
I 3003 2 7+\ 3-79 3 00 -3 27
290-297 \ 300-321
c
m
c
C
N
0
t
I.
_I
:
I
I
Fig. 6. Deletions within the linker between the p53 DNA binding and
tetramerization domains confer novel DNA binding properties to p53.
In vitro translated deletion mutants of wild-type p53 were assayed for
binding to 32P-labeled oligonucleotides BC or BC.S21. The former
DNA contains contiguous half-sites; the latter contains a 21 nucleotide
spacer between the two half-sites. Where indicated the p53 deletion
mutants were translated separately and then coincubated with DNA.
All mutants were examined at equimolar concentrations, except for
p53A300-321 and p53A3-79A300-321, which were incubated at
5-fold lower concentrations in the series of experiments involving
oligonucleotide BC.
Aberrant p53-DNA complexes induced by a short
linker between the DNA binding and
tetramerization domains
Deletion of residues 324-332, 352-393 and 300-327
abolished binding of human p53
BC.V4A (Figure IA). To evaluate these deletion mutants
further, we examined their binding to oligonucleotide BC,
which contains the optimal p53 binding site. The former
two deletion mutants failed to bind this DNA (data not
shown). In contrast, p53A300-327 bound oligonucleotide
BC and, unexpectedly, formed two types of DNA com-
plexes. One of these complexes migrated aberrantly slowly
as compared with the DNA complexes of p53A300-321
(Figure 6); the latter complexes essentially comigrate with
the complexes of wild-type p53 or any other C-terminal
deletion mutant we have constructed (Figure IA).
Migration of protein-DNA complexes depends in part
on their molecular size (Hope and Struhl, 1987). We
therefore examined whether slowly migrating complexes
of p53A300-327 contain more than one p53 tetramer. To
validate our approach we first demonstrated that DNA
complexes of p53A300-321 contained one p53 tetramer.
The DNA complexes ofp53A300-321 and its N-terminally
truncated variant (p53A3-79A300-321) migrated distinctly
(Figure 6). When cotranslated, p53A300-321 and p53A3-
79A300-321 formed heterotetramers which could be
resolved by electrophoresis (data not shown, but see
Figure 1B). However, when translated separately and then
coincubated with DNA, no novel complexes were observed
(Figure 6), suggesting that tetramers, once formed, did
not exchange subunits and that one p53 tetramer was
bound to DNA. We next focused on p53A300-327. Its N-
terminally truncated variant, p53A3-79A300-327, formed
aberrantly slowly migrating complexes with DNA as
compared with p53A3-79A300-321 (Figure 6). Coincuba-
tion of p53A300-327 and p53A3-79A300-327 translated
to oligonucleotide
separately with DNA led to one novel complex, suggesting
that the slowly migrating DNA complexes of p53A300-
327 contain two p53 tetramers (Figure 6).
The aberrant DNA binding properties of p53A300-327,
as compared with p53A300-321, can be attributed to
deletion of residues 322-327 or to shortening of the linker
between the DNA binding and tetramerization domains.
To distinguish between these possibilites we examined
additional deletion mutants involving the region between
the C-terminal boundary of the DNA binding domain
(residue 289) and the tetramerization domain. The DNA
complexes of p53A290-297 migrated like those of wild-
type p53
or p53A300-321.
297A300-321 formed slowly migrating DNA complexes
(Figure 6). Thus, single deletions of residues 290-297 or
300-321 did not confer aberrant DNA binding, while the
double deletion did, suggesting that a short linker between
the DNA binding and tetramerization domains, rather than
deletion of specific residues correlates with aberrant
binding.
A
shortlinkerbetween
tetramerization domains might restrict the ability of the
p53 DNA binding domains to align with contiguous
pentanucleotides, such as those present in oligonucleotide
BC. According to this model, if one p53 tetramer occupied
only one half-site on the DNA, then a second p53 tetramer
could occupy the second half-site, thus leading to DNA
complexes containing two tetramers. One prediction of
this model might be that a spacer between the two DNA
half-sites, could allow both half-sites to be recognized by
the same p53 tetramer. To test this prediction we generated
a variant of oligonucleotide BC, which contains 21 non-
specific nucleotides between the half-sites. The length of
the spacer was chosen to maintain the p53 half-sites on
the same side of the DNA molecule. Deletion mutants
p53A300-321, p53A300-327 and p53A290-297A300-321
all bound to oligonucleotide BC.S21. Furthermore, their
DNA complexes comigrated (Figure 6). To determine how
many p53 tetramers are present in the complexes of
p53A300-327 with BC.S21 DNA, we translated separately
p53A300-327 and p53A3-79A300-327. Upon coincuba-
tion with BC.S21 DNA no novel p53-DNA complexes
were observed evenat long exposures
suggesting that separating the half-sites on the DNA
by 21 nucleotides allows one p53A300-327 tetramer to
recognize both half-sites.
In
contrast,
p53A290-
the DNA
binding
and
(Figure6),
Efficient binding of wild-type p53 to DNA with a
21 nucleotide spacer between the half-sites
Wild-type p53 does not bind suboptimal DNA sites
efficiently in vitro, unless induced to switch conformation
to a state with high affinity for DNA (R state). The
conformational switch involves a change in p53 quaternary
structure, but not in subunit stoichiometry (Halazonetis
and Kandil, 1993; Halazonetis et al., 1993). Prompted by
the
ability
of
deletion
mutants p53A300-327
p53A290-297A300-321 to bind a DNA site with a spacer
between the half-sites, we wondered if a similar spacer
would allow wild-type p53 to recognize suboptimal DNA
sites. Two variants of the suboptimal BC.V4A DNA
site, BC.V4A.S10 and BC.V4A.S21, were generated by
inserting
10 and 21nucleotide spacers, respectively,
between the half-sites of BC.V4A. The lengths of the
and
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