Folding and misfolding mechanisms of the p53 DNA binding domain at physiological temperature

p53 modulates a large number of cellular response pathways and is critical for the prevention of cancer. Wild-type p53, as well as tumorigenic mutants, exhibits the singular property of spontaneously losing DNA binding activity at 37 degrees C. To understand the molecular basis for this effect, we examine the folding mechanism of the p53 DNA binding domain (DBD) at elevated temperatures. Folding kinetics do not change appreciably from 5 degrees C to 35 degrees C. DBD therefore folds by the same two-channel mechanism at physiological temperature as it does at 10 degrees C. Unfolding rates, however, accelerate by 10,000-fold. Elevated temperatures thus dramatically increase the frequency of cycling between folded and unfolded states. The results suggest that function is lost because a fraction of molecules become trapped in misfolded conformations with each folding-unfolding cycle. In addition, at 37 degrees C, the equilibrium stabilities of the off-pathway species are predicted to rival that of the native state, particularly in the case of destabilized mutants. We propose that it is the presence of these misfolded species, which can aggregate in vitro and may be degraded in the cell, that leads to p53 inactivation.     

Kinetic partitioning during folding of the p53 DNA binding domain

The DNA-binding domain (DBD) of wild-type p53 loses DNA binding activity spontaneously at 37 degrees C in vitro, despite being thermodynamically stable at this temperature. We test the hypothesis that this property is due to kinetic misfolding of DBD. Interrupted folding experiments and chevron analysis show that native molecules are formed via four tracks (a-d) under strongly native conditions. Folding half-lives of tracks a-d are 7.8 seconds, 50 seconds, 5.3 minutes and more than five hours, respectively, in 0.3M urea (10 degrees C). Approximately equal fractions of molecules fold through each track in zero denaturant, but above 2.0M urea approximately 90% fold via track c. A kinetic mechanism consisting of two parallel folding channels (fast and slow) is proposed. Each channel populates an on-pathway intermediate that can misfold to form an aggregation-prone, dead-end species. Track a represents direct folding through the fast channel. Track b proceeds through the fast channel but via the off-pathway state. Track c corresponds to folding via the slow channel, primarily through the off-pathway state. Track d proceeds by way of an even slower, uncharacterized route. We postulate that activity loss is caused by partitioning to the slower tracks, and that structural unfolding limits this process. In support of this view, tumorigenic hot-spot mutants G245S, R249S and R282Q accelerate unfolding rates but have no affect on folding kinetics. We suggest that these and other destabilizing mutants facilitate loss of p53 function by causing DBD to cycle unusually rapidly between folded and unfolded states. A significant fraction of DBD molecules become effectively trapped in a non-functional state with each unfolding-folding cycle.     

Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants.

p53 is a conformationally flexible sequence-specific DNA binding protein mutated in many human tumors. To understand why the mutant p53 proteins associated with human tumors fail to bind DNA, we mapped the DNA binding domain of wild-type p53 and examined its regulation by changes in the protein conformation. Using site-directed mutagenesis, residues 90-286 of mouse p53 were shown to form the sequence-specific DNA binding domain. Two highly conserved regions within this domain, regions IV and V, were implicated in contacting DNA. Wild-type p53 bound DNA as a tetramer, each subunit recognizing five nucleotides of the 20 nucleotide-long DNA site. Conformational shifts of the oligomerization domain propagated to the tetrameric DNA binding domain, regulating DNA binding activity, but did not affect the subunit stoichiometry of wild-type p53 oligomers. Interestingly, conformational shifts could also be propagated within certain p53 mutants, rescuing DNA binding. One of these mutants was the mouse equivalent of human histidine 273, which is frequently associated with human tumors.     

Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2

Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.     

Generation of monospecific peptide antibodies to the DNA binding domain of p53

The DNA binding domain (DBD) is the most mutated region of p53 in tumors and has proven to be relatively resistant to the generation of specific antibodies. Template assembled synthetic peptide (TASP) synthesis of a peptide derived from the DBD creates a highly immunogenic molecule without the need for large carriers such as keyhole limpet hemocyanin (KLH). In addition, a rapid means of generating monoclonal antibodies can be achieved through immunization in conjunction with ABL/MYC retrovirus injection into recipient mice. In this paper, we demonstrate that an antibody generated by this means, KH2, reacts specifically with the DBD of p53. To date, this is the first example of a peptide immunogen used successfully in ABL/MYC monoclonal antibody production. KH2 is also the first example of a monospecific antibody that directly binds to and, by definition, assumes the conformation of the DNA binding region of p53.     

Modulation of binding of DNA to the C-terminal domain of p53 by acetylation

The binding of nonspecific DNA to the C-terminal negative regulatory domain (CTD) of p53 modulates its activity. The CTD is a natively unfolded region, which is subject to acetylation and phosphorylation at several residues as part of control. To measure the effect of covalent modification on binding to DNA, we synthesized a series of fluorescein-labeled CTD peptides with single and multiple acetylations at lysine residues that we had identified by NMR as making contact with DNA, and developed an analytical ultracentrifugation method to study their binding to DNA. Binding depended on ionic strength, indicating an electrostatic contribution. Monoacetylation weakened DNA binding at physiological ionic strength 2- to 3-fold, diacetylations resulted in further 2- to 3-fold decrease in the affinity, and tri- and tetraacetylations rendered DNA binding undetectable. Phosphorylation at S392 did not affect DNA binding. NMR spectroscopy showed binding to DNA did not induce significant structure into CTD, apart possibly from local helix formation.     

Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide

MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein. Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain. We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide. Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria. To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain. To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide. The advantages of our new construct are high yield and easy purification of the MDM2 protein.     

A coproofreading Zn(2+)-dependent exonuclease within a bacterial replicase

The proofreading exonucleases of all DNA replicases contain acidic residues that chelate two Mg(2+) ions that participate in catalysis. DNA polymerase III holoenzymes contain their proofreading activity in a separate subunit, epsilon, which binds the polymerase subunit, alpha, through alpha's N-terminal php domain. Here we demonstrate that the alpha php domain contains a novel Zn(2+)-dependent 3' --> 5' exonuclease that preferentially removes mispaired nucleotides, providing the first example of a coediting nuclease.     

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2)

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.     

The MDMX acidic domain competes with the p53 transactivation domain for MDM2 N-terminal domain binding

The tumor suppressor protein p53 governs many cellular pathways to control genome integrity, metabolic homeostasis, and cell viability. The critical roles of p53 highlight the importance of proper control over p53 in maintaining normal cellular function, with the negative regulators MDM2 and MDMX playing central roles in regulating p53 activity. The interaction between p53 and either MDM2 or MDMX involves the p53 transactivation domain (p53TD) and the N-terminal domains (NTD) of MDM2 or MDMX. Recently, the acidic domain (AD) of MDMX was found to bind to its own NTD, inhibiting the p53-MDMX interaction. Given the established structural and functional similarity between the MDM2 and MDMX NTDs, we hypothesized that the MDMX AD would also directly bind to MDM2 NTD to inhibit p53-MDM2 interaction. Through solution-state nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that the MDMX AD can indeed directly interact with the MDM2 NTD and, as a result, can compete for p53 binding. The MDMX AD is thus able to serve as a regulatory domain to inhibit the MDM2-p53 interaction and may also play a direct role in p53 activation.