(Received for publication, June 14, 1995; and in revised form, October 5, 1995)
From the
Conformational stability is a prerequisite for the physiological activity of the tumor suppressor protein p53. p53 protein can be allosterically activated for DNA binding by phosphorylation or through noncovalent interaction with proteins such as DnaK, the Escherichia coli homologue of the heat shock protein Hsp70. We present in vitro evidence for a rapid temperature-dependent change in the conformation and tetrameric nature of wild-type p53 upon incubation at 37 °C, which correlates with a permanent loss in DNA binding activity. We show that p53 is allosterically regulated for stabilization of the wild-type conformation and DNA binding activity at 37 °C by binding of two classes of ligands to regulatory sites on the N and C terminus of the molecule through which an intrinsic instability of p53 is neutralized. Deletion of the domain conferring instability at the C terminus is sufficient to confer enhanced stability to the total protein. DnaK binding to the C terminus can profoundly protect p53 at 37 °C from a temperature-dependent loss of the DNA binding activity but does not renature or activate denatured p53. In contrast, another activator of the DNA binding activity of latent p53, the monoclonal antibody PAb421, which also interacts with the C terminus of the protein, is not able to protect p53 from thermal denaturation. Two monoclonal antibodies to the N terminus of p53, PAb1801 and DO-1, do not activate the latent DNA binding function of p53 but can protect the p53 wild-type conformation at 37 °C. Thus, activation of the DNA binding function of p53 is not synonymous with protection from thermal denaturation, and therefore, both of these pathways may be used in cells to control the physiological activity of p53. The protection of p53 conformation from heat denaturation by interacting proteins suggests a novel mechanism by which p53 function could be regulated in vivo.
Wild-type p53 is a nuclear phosphoprotein of 393 amino acids
(for reviews see Prives(1994) and Lane(1994)) with well characterized
domains contributing to its function and regulation. The N-terminal
domain functions as a transcriptional activator (Fields and Jang, 1990;
Raycroft et al., 1990; Farmer et al., 1992; Unger et al., 1992), and the C terminus has been shown to contain a
tetramerization domain (Shaulian et al., 1992; Clore et
al., 1994; Waterman et al., 1995) and to regulate the
specific DNA binding function of p53 (Hupp et al., 1992, 1993,
1995). The core region (residues 90-290) consists of the
sequence-specific DNA binding domain (Bargonetti et al., 1993;
Pavletich et al., 1993; Wang et al., 1993) and
contains four of the five regions that are evolutionarily conserved
among vertebrates (Soussi et al., 1990). The specific p53 DNA
binding element is found to be a repeat of 10 base pairs
(RRRC(A/T)(T/A)GYYY) where each monomer of tetrameric p53
can bind to five base pairs (Kern et al., 1991; El-Deiry et al., 1992; Halazonetis and Kandil, 1993).
Co-crystallization of the core domain with a DNA duplex containing a
half consensus site revealed the critical amino acids for DNA binding
(Cho et al., 1994). Binding of p53 to the consensus element
can drive the transcription of reporter genes (Funk et al.,
1992), and the element has been shown to be present in promotor or
intron regions of several genes including p21
(El-Deiry et al., 1993; Harper et al., 1993), gadd45 (Kastan et al., 1992), the muscle creatine
kinase gene (Weintraub et al., 1991; Zambetti et al.,
1992), and the mdm2 gene (Barak et al., 1993).
The importance of this core region for the function of p53 is underlined by the fact that of more than 2000 described mutations of the p53 gene, the majority are point mutations clustered in the DNA binding domain (Pavletich et al., 1993; Cho et al., 1994). Many of these point mutations can be correlated with a loss of the wild-type conformation as detected by the loss of reactivity to the monoclonal antibodies PAb1620, specific for murine and human p53 (Milner et al., 1987), and the mouse-specific PAb246 (Yewdell et al., 1986) or the gain of reactivity to the mouse-specific monoclonal antibody PAb240 (Gannon et al., 1990; Stephen and Lane, 1992).
Understanding the regulation of p53 conformation and biochemical activity is essential for understanding how cells use the p53 pathway to regulate growth control. To date, p53 protein is known to be activated for sequence-specific DNA binding by covalent modification (i.e. phosphorylation of its negative regulatory domain) or by noncovalent modification (binding by the monoclonal antibody PAb421 or bacterial Hsp70) (Delphin and Baudier, 1994; Hupp and Lane, 1994a, 1994b). The mechanism whereby an antibody activates p53 has been studied most extensively; activation occurs through a concerted mechanism in which the tetrameric nature of p53 is maintained (Hupp and Lane, 1994a). In addition, activation by casein kinase II or protein kinase C is reversed by monoclonal antibodies or phosphatases, respectively, indicating that activation of p53 is a reversible process possibly subject to refined metabolic control (Hupp and Lane, 1994a; Takenaka et al., 1995).
Here, we demonstrate that the conformation of wild-type p53 is very temperature-sensitive. The loss of the PAb1620-reactive epitope correlates with a loss of the tetrameric nature of p53 and with a loss of the p53 DNA binding activity. We find that the Escherichia coli homologue of Hsp70, DnaK, and two human Hsp70 proteins can protect p53 DNA binding at 37 °C. Specific monoclonal antibodies to the N terminus of p53 can also stabilize the PAb1620-positive conformation and DNA binding activity of p53 at 37 °C.
Recombinant human Hsp70 (p72) and Hsc70 (p73), purified from E. coli, were obtained from Stress Gen, Biotechnologies Corp.
Figure 1: Thermal instability of wild-type p53 conformation determined by two-site ELISA. Varying amounts of wild-type p53 incubated for 10 min at the indicated temperatures were added to microtiter well plates precoated with the indicated conformationally specific monoclonal antibody. p53 bound to the monoclonal antibody was detected with the rabbit polyclonal antiserum CM-1 and swine anti-rabbit IgG conjugated to horseradish peroxidase. The assay was carried out at 21 °C. Data is shown for 50 ng of recombinant human p53 expressed in E. coli (A, B, and D) and recombinant murine p53 expressed in Sf9 insect cells (C).
Figure 2: Effect of temperature on oligomeric structure of p53. The differences in sedimentation of 1 µg human p53 expressed in Sf9 insect cells, incubated for 1 h at 4 or 37 °C respectively, was determined by a 5-40% sucrose gradient at 4 °C and subsequent analysis of the collected fractions by a two-site ELISA using the monoclonal antibody DO-1 to capture p53 on the plate and the polyclonal antiserum CM-1 for detection. The position of tetrameric p53 (6 S) is indicated by the arrow.
Figure 3: Effect of temperature on DNA binding activity of p53. 150 ng of human p53 expressed in E. coli were incubated for 5 min at the indicated temperatures and subsequently subjected to a DNA binding assay including the monoclonal antibody PAb421 performed at 4 °C (A) or a Western blot detecting p53 with the monoclonal antibodies PAb421, DO-1 and PAb240 by ECL (Amersham Corp.) (B).
Figure 4:
DNA binding of different forms of p53
incubated at 37 °C. DNA binding assays were performed on 150 ng of
p53 preincubated at 37 °C for 5-60 min with p53 produced in E. coli, which was activated with 100 ng PAb421 after the
incubation at 37 °C (A), or p53 expressed in Sf9 insect
cells (B). C, the C-terminal 30 mutant produced
in insect cells was subjected to the same incubation at 37 °C
before analysis by DNA binding assay.
Figure 5: Effect of heat shock proteins on the p53 DNA binding activity at 37 °C. A, human p53 expressed in bacteria (1 µM) and DnaK (15 µM) were incubated at 37 °C as indicated and subsequently analyzed for DNA binding activity. B, p53 was incubated alone at 37 °C for the indicated times and subsequently incubated with DnaK at 30 °C for 30 min. C, 1 µM insect cell-produced p53 was analyzed for DNA binding after incubation at 4 °C and 60 min at 37 °C including bovine serum albumin (3.7 µM), human Hsp70 (p72) (3.4 µM), and human Hsc70 (p73) (3.4 µM), respectively.
Figure 6: Stabilization of p53 at 37 °C by monoclonal antibodies. A, after co-incubation at 37 °C for 5-60 min with the indicated monoclonal antibodies, p53 (1 µM) was analyzed in a DNA binding assay; insect cell-produced p53 was incubated with 0.6 µM DO-1 (a) and 0.6 µM PAb1801 (b). p53 produced in bacteria was incubated with 0.6 µM PAb421 (c). B, quantitative analysis of maximal stabilization by DO-2, DO-1, PAb1801, and PAb421 of the DNA binding activity of p53 at 37 °C in relation to activity at 4 °C. The error bars represent the standard error of the mean. C, stabilization of DNA binding of p53 produced in insect cells (1 µM) at 37 °C by increasing concentrations of PAb1801 quantified by PhosphorImager. D, increasing amounts of bacterially produced p53 were analyzed by a two-site ELISA captured by PAb1620 and detected with CM-1 as in ``Materials and Methods.'' p53 and PAb1801 (0.6 µM) were incubated at 37 °C for 30 and 60 min compared with 21 °C.
We determined the maximal protection achieved by the respective antibodies under optimized conditions after 60 min of incubation at 37 °C expressed as the relative percentage of DNA binding compared with binding at 4 °C (Fig. 6B). PAb1801 was the most potent stabilizing agent by protecting up to 77% of the DNA binding activity of p53. Interestingly, although DO-1 seemed to protect p53 consistently over the 60 min observed (Fig. 6A, part a), the effect was limited to only 39% of the total protein activity. Another antibody to the N terminus, DO-2, binding to amino acids 6-15 (Legros et al., 1994), was also found to have some stabilizing properties (16%), whereas the effect of PAb421 was comparatively minor (3%) after 60 min at 37 °C. We characterized the kinetics of stabilization by titrating increasing amounts of PAb1801 into a constant amount of p53 and then incubated the mix for 60 min at 37 °C (Fig. 6C). Lower concentrations of 0.005-0.05 µM PAb1801 did not stabilize the DNA binding activity significantly, but higher concentrations led to a dramatically increased stabilizing effect resulting in a sigmoidal binding curve, which is characteristic for an allosterically regulated reaction. Evidence for the notion that stabilization requires binding of PAb1801 to all four epitopes on tetrameric p53 comes from gel shift assays with a titration of PAb1801 at 4 °C. At PAb1801 concentrations at which only one antibody molecule is bound per tetramer, there is no protection of the DNA binding of p53 at 37 °C (data not shown).
In light of the stabilizing properties displayed by the N-terminal binding antibodies in a DNA binding assay, we investigated whether antibodies of this sort could also stabilize the native conformation as assayed by PAb1620 reactivity. The conformational changes of p53 were measured by ELISA as described under ``Materials and Methods.'' p53 was incubated at 37 °C for 30 and 60 min before being captured with PAb1620 and detected by CM-1. When p53 was co-incubated with the monoclonal antibody PAb1801 at 37 °C, PAb1801 was able to protect the PAb1620-positive conformation (Fig. 6D).
Incubations at physiologically relevant temperatures affect p53 at the structural and functional level. The results reported here demonstrate that wild-type p53 is a highly temperature-sensitive protein in two respects. On a structural level, the protein undergoes irreversible conformational changes after short incubations (10 min) at temperatures in a range from 37 to 45 °C by losing its wild-type (PAb1620-positive) conformation. Because the assay for detecting the epitope changes of p53 is carried out at room temperature, the temperature-dependent differences are due to the initial heat treatment of 10 min, since the protein was theoretically allowed to refold during the progress of the ELISA. For this reason we believe that the monitored changes are irreversible and that there is no spontaneous refolding. Incubation at 37 °C abolishes the tetrameric nature of p53 and leads to formation of higher molecular weight aggregates. At a functional level, p53 is permanently inactivated for DNA binding by an incubation of 5 min at 45 °C, which confirms the PAb1620-positive conformation as a prerequisite for DNA binding (Halazonetis et al., 1993). Longer incubations at 37 °C also result in a loss of DNA binding activity. This thermoinstability is seen for both human and murine wild-type p53 expressed in eukaryotic and prokaryotic systems.
p53 has been shown to be a conformationally dynamic protein in vivo and in vitro (Gannon et al., 1990;
Milner and Medcalf, 1990). These results suggest that the conformation
of the central core domain of p53 is essential for its biological
activity. A loss of the PAb1620-reactive epitope has been reported for
quiescent cells upon addition of fresh medium and reentry into the cell
cycle (Milner and Watson, 1990). Interestingly, we show that
PAb1620-negative p53 cannot be activated by PAb421 or DnaK for DNA
binding. Thus it is no longer subject to the activation pathway (Fig. 7A), which is believed to contribute to the
p53-mediated cell cycle arrest via a transcriptional activation of
target genes. Thus one can speculate that a possible physiological role
of p53 is to allow cell proliferation due to loss
of p53's physiological activity.
Figure 7: A, activation for DNA binding and thermostability of p53 are two separate pathways. The activation for DNA binding of latent (1620-positive) p53 by kinases (protein kinase C and casein kinase II) can be mimicked by the monoclonal antibody PAb421 but not by PAb1801. Protein phosphatases (PP1 and PP2A) and monoclonal antibodies (ICA-9) can reverse the activation. Both forms of p53, activated and latent, are prone to thermal denaturation as monitored by a loss of the 1620-positive conformation, but activation dos not confer significant stability to the protein. Although denatured p53 is drawn as monomers, it corresponds to aggregates of higher molecular weight (see Fig. 2). Binding of monoclonal antibodies to the N terminus (PAb1801) confers conformational stability to both forms of the protein, which is not achieved by PAb421. The interaction of the bacterial Hsp70 homologue DnaK with p53 is of a different nature, since it can act on both pathways: activation and protection from denaturation. B, model for the allosteric regulation of the thermostability of p53 DNA binding and conformation. p53 loses its wild-type conformation in the central core of the molecule in a temperature-dependent manner, with the N and C termini contributing to its instability. Deletion of a domain conferring instability within the last 30 amino acids of the C terminus or binding of DnaK within that region thermally stabilizes the core of the protein. In the same way, binding of monoclonal antibodies to specific domains at the N terminus also leads to different degrees of thermostability of p53.
Investigations of the conformational stability of globular proteins (such as ribonuclease T1 and barnase) have shown that several factors account for stability: temperature, disulfide bonds, amino acid sequence, salt concentrations, and pH. Higher as well as lower temperatures can decrease protein stability and lead to unfolding (for review see Pace(1990)). Proteins show only a small free energy of stabilization as compared with the total molecular energy. As a result, molecular adaptations to extreme physical conditions like temperature require only marginal alterations of the intermolecular interactions and packing density. Enhanced stability can be achieved by mutations at the protein level or binding of extrinsic factors such as ions, co-factors or specific ligands in vivo and in vitro. We demonstrate here that the binding of specific ligands as well as the deletion of destabilizing domains can confer stability to the wild-type conformation of p53.
Phosphorylation of p53 at the C terminus allosterically activates
the latent DNA binding activity of p53, possibly by removing the
C-terminal domain from a site of interaction in the central part of the
molecule (Hupp et al., 1995). Similarly, deletion of the
C-terminal 30 amino acids (30) leads to constitutive activation
(Hupp et al., 1992). Since this allosteric activation could
thereby confer some stability to the molecule, we compared the
thermostability of phosphorylated p53 produced in insect cells with
unphosphorylated p53 expressed in bacteria and found no significant
stabilization due to the phosphorylation. Surprisingly,
30 protein
shows a dramatically enhanced intrinsic stability to temperature,
suggesting that a region at the C terminus confers some instability to
p53.
To determine the role of
allosteric activation in the stabilization of p53 at 37 °C, we used
the monoclonal antibody PAb421, which activates p53 for DNA binding
(Hupp et al., 1992). PAb421 is not able to activate denatured
p53 (Fig. 4A), nor is it able to protect p53 from
denaturation (Fig. 6A, after a 15-min incubation at 37
°C), clearly showing that activation and conformational
stabilization are two different pathways (Fig. 7A). We
have identified a region at the N terminus (amino acids 46-55) of
p53 that stabilizes p53 dramatically (60-70%) when bound by the
antibody PAb1801. Indeed, the ability of antibodies to confer stability
to p53 increases with antibodies binding in closer proximity to the
PAb1801 epitope (Fig. 7B). One interpretation of these
results is that the epitope recognized by PAb1801 confers some
instability to p53, which can be neutralized by the binding of the
antibody. A deletion of this region should therefore result in a gain
of stability, as it is seen for 30. Indeed, a deletion at the N
terminus of 102 N-terminal amino acids including the PAb1801 epitope
renders p53 more stable to temperature (data not shown). We demonstrate
that the ability of PAb1801 to stabilize p53 DNA binding at 37 °C
increases dramatically with increasing concentrations and rapidly
reaches a plateau, resulting in a sigmoidal binding curve (Fig. 6C). From a titration of PAb1801 at 4 °C it
was evident that at nonprotective concentrations of PAb1801 only one
antibody was bound per tetramer, since p53 was only partially
supershifted to an intermediate form (data not shown). Thus, efficient
stabilization only takes place when all subunits of the tetrameric p53
are bound by two bivalent antibody molecules. Fab fragment analysis
showed that the bivalency of the antibody was not necessary for the
stabilization, although the effect was somewhat weaker, but significant
stabilization was achieved only with all four Fab fragments bound to
p53 (data not shown). The stabilization effect of PAb1801 seems to be
mediated by a protection of the wild-type conformation (Fig. 6D). Since p53 denatures in a rapid manner at 37
°C, the antibody concentration seems to be critical to protect p53.
Over the time observed (up to 60 min), the population active for DNA
binding remains unchanged (Fig. 6A, parts a and b), although it might only represent a small
percentage of the total DNA binding activity (e.g. 39%
stabilization by DO-1). A deletion of the C terminus of p53, while
substantially delaying the inactivation of p53 at 37 °C, is less
effective in protecting the activity of p53 than the binding of
antibody or DnaK (Fig. 4C).
Taken together, our data indicate that p53 can be allosterically regulated for stabilization of the wild-type conformation and DNA binding activity at 37 °C by binding of two classes of ligands (e.g. PAb1801 and DnaK) to regulatory sites on both ends of the molecule, outside of the DNA binding domain, by which an intrinsic instability of p53 is neutralized (Fig. 7B). The deletion of motifs conferring instability either at the C or N terminus is enough to confer enhanced stability to the total protein as well as the binding of only one specific ligand. Hence we provide an assay to identify cellular factors that regulate p53 stability in vivo. We can clearly show that activation for DNA binding and stabilization are two separate pathways (Fig. 7A). In this model, misfolded protein (PAb1620-negative) would be targeted for rapid degradation by cellular proteolytic pathways. Monoclonal antibodies can often substitute for essential ligands or regulatory factors; binding of PAb421 to p53 can substitute for activation by protein kinase C and casein kinase II (Hupp and Lane, 1994). PAb1801 (and DO-1) could possibly mimic a class of cellular proteins that regulate p53 stability by interacting with the N terminus. It is of great interest that the DO-1 antibody interacts with critical amino acids in the N terminus of the protein required for binding the mdm2 oncogene and the transcriptional co-activators TAFII40 and TAFII60 (Picksley et al., 1994; Thut et al., 1995). Interestingly, the WT1 gene product, which binds to the 70 N-terminal amino acids of p53, has recently been shown to stabilize p53, resulting in increased steady-state levels of p53, and it partially protects against papillomavirus E6-mediated degradation of p53, which correlates to the presence of high levels of p53 protein in primary Wilms' tumors (Maheswaran et al., 1995). Regulation of the thermostability of p53 may represent a new mechanism to control the biological activity of this important tumor suppressor gene product.