The p73 DNA binding domain displays enhanced stability relative to its homologue, the tumor suppressor p53, and exhibits cooperative DNA binding

The p53 protein family is involved in the control of an intricate network of genes implicated in cell cycle, through to germ line integrity and development. Although the role of p53 is well-established, the intrinsic nature of its homologue p73 has yet to be fully elucidated. Here, the biochemical characterization and homology-based modeling of the p73 protein is presented and the implications for its function(s) examined. The DNA binding domains (DBDs) of p53, p63, and p73 bind to the specific target site of a 30-mer gadd45 dsDNA, as tested by EMSA. The monomeric DBDs bind cooperatively forming tetrameric complexes. However, a larger construct consisting of p73 DBD plus TET domain (p73 CT) and the corresponding p53 DBD plus TET domain (p53 CT) bind gadd45 differently than the respective DBDs. Significantly, p73 DBD exhibited enhanced thermodynamic stability relative to the p53 DBD but not compared to p63 DBD as shown by DSC, CD, and equilibrium unfolding. The p73 CT is less stable than p73 DBD. The modeling data show distinct electrostatic surfaces of p73 and p53 dimers when bound to DNA. Specifically, the p73 surface is less complementary for DNA binding, which may account for the differences in affinity and specificity for p53 REs. These stability and DNA binding data for p73 in vitro enhance and complement our understanding of the role of the p73 protein in vivo and could be exploited in designing strategies for cancer therapy in places where p53 is mutated.     

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.     

Pliable DNA conformation of response elements bound to transcription factor p63

We show that changes in the nucleotide sequence alter the DNA conformation in the crystal structures of p63 DNA-binding domain (p63DBD) bound to its response element. The conformation of a 22-bp canonical response element containing an AT spacer between the two half-sites is unaltered compared with that containing a TA spacer, exhibiting superhelical trajectory. In contrast, a GC spacers abolishes the DNA superhelical trajectory and exhibits less bent DNA, suggesting that increased GC content accompanies increased double helix rigidity. A 19-bp DNA, representing an AT-rich response element with overlapping half-sites, maintains superhelical trajectory and reveals two interacting p63DBD dimers crossing one another at 120°. p63DBD binding assays to response elements of increasing length complement the structural studies. We propose that DNA deformation may affect promoter activity, that the ability of p63DBD to bind to superhelical DNA suggests that it is capable of binding to nucleosomes, and that overlapping response elements may provide a mechanism to distinguish between p63 and p53 promoters.     

Structural evolution of C-terminal domains in the p53 family

The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C-terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they both differ significantly from each other as well as from human p53. CEP-1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N-terminal beta-strand and a C-terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53.     

Folding of Tetrameric p53: Oligomerization and Tumorigenic Mutations Induce Misfolding and Loss of Function

The physiologically active form of p53 consists of a tetramer of four identical 393-amino-acid subunits associated via their tetramerization domains (TDs; residues 325-355). One in two human tumors contains a point mutation in the DNA binding domain (DBD) of p53 (residues 94-312). Most existing studies on the effects of these mutations on p53 structure and function have been carried out on the isolated DBD fragment, which is monomeric. Recent structural evidence, however, suggests that DBDs may interact with each other in full-length tetrameric forms of p53. Here, we investigate the effects of tumorigenic DBD mutations on the folding of p53 in its tetrameric form. We employ the construct consisting of DBD and TD (amino acids 94-360). We characterize the stability and conformational state of the tumorigenic DBD mutants R248Q, R249S, and R282Q using equilibrium denaturation and functional assays. Destabilizing mutations cause DBD to misfold when it is part of the p53 tetramer, but not when it is monomeric. This conformation is populated under moderately destabilizing conditions (10 °C in 2 M urea, and at physiological temperature in the absence of denaturant). Under those same conditions, it is not present in the isolated DBD fragment or in the presence of the TD mutation L344P, which abolishes tetramerization. Misfolding appears to involve intramolecular DBD-DBD association within a single tetrameric molecule. This association is promoted by destabilization of DBD (caused by mutation or elevated temperature) and by the high local DBD concentration enforced by tetramerization of TD. Disrupting the nonnative DBD-DBD interaction or transiently inhibiting tetramerization and allowing p53 to fold as a monomer may be potential strategies for pharmacological intervention in cancer.     

The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.     

R248Q mutation—Beyond p53‐DNA binding

R248 in the DNA binding domain (DBD) of p53 interacts directly with the minor groove of DNA. Earlier nuclear magnetic resonance (NMR) studies indicated that the R248Q mutation resulted in conformation changes in parts of DBD far from the mutation site. However, how information propagates from the mutation site to the rest of the DBD is still not well understood. We performed a series of all‐atom molecular dynamics (MD) simulations to dissect sterics and charge effects of R248 on p53‐DBD conformation: (i) wild‐type p53 DBD; (ii) p53 DBD with an electrically neutral arginine side‐chain; (iii) p53 DBD with R248A; (iv) p53 DBD with R248W; and (v) p53 DBD with R248Q. Our results agree well with experimental observations of global conformational changes induced by the R248Q mutation. Our simulations suggest that both charge‐ and sterics are important in the dynamics of the loop (L3) where the mutation resides. We show that helix 2 (H2) dynamics is altered as a result of a change in the hydrogen bonding partner of D281. In turn, neighboring L1 dynamics is altered: in mutants, L1 predominantly adopts the recessed conformation and is unable to interact with the major groove of DNA. We focused our attention the R248Q mutant that is commonly found in a wide range of cancer and observed changes at the zinc‐binding pocket that might account for the dominant negative effects of R248Q. Furthermore, in our simulations, the S6/S7 turn was more frequently solvent exposed in R248Q, suggesting that there is a greater tendency of R248Q to partially unfold and possibly lead to an increased aggregation propensity. Finally, based on the observations made in our simulations, we propose strategies for the rescue of R248Q mutants. Proteins 2015; 83:2240–2250. © 2015 Wiley Periodicals, Inc.     

Molecular dynamics simulations suggest Thiosemicarbazones can bind p53 cancer mutant R175H

Cancer pathologies are associated with the unfolding and aggregation of most recurring mutations in the DNA Binding Domain (DBD) of p53 that coordinate the destabilization of protein. Substitution at the 175th codon with arginine to histidine (R175H, a mutation of large to small side-chain amino acid) destabilizes the DBD by 3 kcal/mol and triggers breasts, lung cancer, etc. Stabilizing the p53 mutant by small molecules offers an attractive drug-targeted anti-cancer therapy. The thiosemicarbazone (TSC) molecules NPC and DPT are known to act as zinc-metallochaperones to reactivate p53R175H. Here, a combination of LESMD simulations for 10 TSC conformations with a p53R175H receptor, single ligand-protein conformation MD, and ensemble docking with multiple p53R175H conformations observed during simulations is suggested to identify the potential binding site of the target protein in light of their importance for the direct TSC – p53R175H binding. NPC binds mutant R175H in the loop region L2-L3, forming pivotal hydrogen bonds with HIS175, pi?sulfur bonds with TYR163, and pi-alkyl linkages with ARG174 and PRO190, all of which are contiguous to the zinc-binding native site on p53DBD. DPT, on the other hand, was primarily targeting alternative binding sites such as the loop-helix L1/H2 region and the S8 strand. The similar structural characteristics of TSC-bound p53R175H complexes with wild-type p53DBD are thought to be attributable to involved interactions that favour binding free energy contributions of TSC ligands. Our findings may be useful in the identification of novel pockets with druggable properties.     

Aggregation of zinc‐free p53 is inhibited by Hsp90 but not other chaperones

The structured DNA‐binding domain (DBD) of p53 is a well‐known client protein of the chaperone Hsp90. The p53 DBD contains a single zinc ion, coordinated by the side chains of Cys176, His179, Cys238, and Cys242; zinc coordination plays a structural role to stabilize the DBD and is required for its DNA binding. The ambiguous nature of the p53‐Hsp90 interaction, together with the stabilizing role of the zinc in the structure of the DBD, prompted us to examine the interaction of Hsp90 with zinc‐free p53 DBD. NMR spectroscopy and native gel electrophoresis did not show any apparent preference for the interaction of the destabilized zinc‐free form of p53 DBD with Hsp90. Intriguingly, however, at lower protein concentrations, closer to physiological concentrations, the addition of Hsp90, but not other chaperones such as Hsp70, Hsp40, p23, and HOP, appears to slow or prevent the aggregation of zinc‐free p53 DBD. This result suggests that part of the function of the Hsp90‐p53 interaction in the cell may be to stabilize the apoprotein in the absence of zinc.     

Identification of p53 Sequence Elements That Are Required for MDM2-Mediated Nuclear Export

It has been demonstrated that MDM2 can differentially regulate subcellular distribution of p53 and its close structural homologue p73. In contrast to MDM2-mediated p53 nuclear export, p73 accumulates in the nucleus as aggregates that colocalize with MDM2. Distinct distribution patterns of p53 and p73 suggest the existence of unique structural elements in the two homologues that determine their MDM2-mediated relocalization in the cell. Using a series of p53/p73 chimeric proteins, we demonstrate that three regions of p53 are involved in the regulation of MDM2-mediated nuclear export. The DNA binding domain (DBD) is involved in the maintenance of a proper conformation that is required for functional activity of the nuclear export sequence (NES) of p53. The extreme C terminus of p53 harbors several lysine residues whose ubiquitination by MDM2 appears to be the initial event in p53 nuclear export, as evidenced by the impaired nucleocytoplasmic shuttling of p53 mutants bearing simultaneous substitutions of lysines 370, 372, 373, 381, 382, and 386 to arginines (6KR) or alanines (6KA). Finally, the region between the DBD and the oligomerization domain of p53, specifically lysine 305, also plays a critical role in fully revealing p53NES. We conclude that MDM2-mediated nuclear export of p53 depends on a series of ubiquitination-induced conformational changes in the p53 molecule that lead to the activation of p53NES. In addition, we demonstrate that the p53NES may be activated without necessarily disrupting the p53 tetramer.     

Dimerization of the core domain of the p53 family: A computational study

Computational models reveal the structural origins of cooperativity in the association of the DNA binding domains (DBD) of p53 (and its two homologues p63 and p73) with consensus DNA. In agreement with experiments they show that cooperativity, as defined by sequential binding of monomers to DNA is strong for p53 and weak for homologues p63 and p73. Computations also suggest that cooperativity can arise from the dimerization of the DBD prior to binding the DNA for all 3 family members. Dimerization between the DBDs is driven by packing interactions originating in residues of helix H1 and loop L3, while DNA binding itself is dominated by local and global electrostatics. Calculations further suggest that low affinity oligomerization of the p53 DBD can precede the oligomerization of the tetramerization domain (TD). During synthesis of multiple chains on the polysome, this may increase fidelity by reducing the possibility of the highly hydrophobic TD from nonspecific aggregation. Mutations have been suggested to test these findings.     

p53 mutants without a functional tetramerisation domain are not oncogenic

p53 is altered in about 50 % of cancers. Most of the p53 mutants have lost the wild-type tumour suppressor activity but show oncogenic properties. The majority of the p53 alterations are missense mutations of residues located in its DNA binding domain (DBD). Only a few mutations concern residues in its tetramerisation domain (TD). However, the study of mutant proteins identified in tumors that do not form tetramers has shown that they have lost the wild-type activity like most of the p53 DBD mutants. Here, we show that two of such mutant proteins, Arg342Pro and Leu344Pro are not dominant negative and do not stimulate the expression of a reporter gene under the control of the multi-drug resistance gene-1 (MDR-1). This suggests that to be oncogenic, p53 mutants need to form tetramers. Accordingly, the dominant negative effect and the ability of a tetrameric mutant protein, Asp281Gly, to stimulate the MDR-1 promoter are abolished when its TD is rendered non-functional by the mutation of leucine 344 to a proline residue. These results suggest that mutations in the TD, are less selected in tumors than mutations in the DBD because they do not lead to oncogenic proteins.     

Zn(2+)-dependent misfolding of the p53 DNA binding domain

The DNA binding domain (DBD) of p53 folds by a complex mechanism that involves parallel pathways and multiple intermediates, both on- and off-pathway. This heterogeneity renders DBD particularly susceptible to misfolding and aggregation. The origins of parallel folding mechanisms are not well understood. DBD folding heterogeneity may be caused by the presence of the single bound Zn2+. To test that hypothesis, we carried out kinetic folding studies of DBD in its Zn2+-free form (apoDBD) and in the presence of various concentrations of free Zn2+ and the Zn2+-nitrilotriacetate (NTA) complex. Folding kinetics of apoDBD and DBD are similar, although apoDBD folds faster than DBD at some urea concentrations. The principle consequence of Zn2+ removal is to accelerate unfolding and simplify it from two exponential phases to one. Metal binding interactions are therefore not responsible for the observed complexity of the folding reaction. A slight stoichiometric excess of free Zn2+ arrests folding and traps the protein in a misfolded state in which Zn2+ is bound to nonphysiological ligands. Folding can be rescued by providing metal ions in the form of the NTA-Zn2+ complex, which simultaneously protects against misligation and provides a source of Zn2+ for regenerating the functional protein. This chemical metallochaperone strategy may be an effective means for improving folding efficiency of other metal binding proteins. The findings suggest that, in vivo, DBD must fold in an environment where free Zn2+ concentration is low and its bioavailability is carefully regulated by cellular metallochaperones.     

Structure,function, and aggregation of the zinc-free form of the p53 DNA binding domain

The p53 DNA binding domain (DBD) contains a single bound zinc ion that is essential for activity. Zinc remains bound to wild-type DBD at temperatures below 30 degrees C; however, it rapidly dissociates at physiological temperature. The resulting zinc-free protein (apoDBD) is folded and stable. NMR spectra reveal that the DNA binding surface is altered in the absence of Zn(2+). Fluorescence anisotropy studies show that Zn(2+) removal abolishes site-specific DNA binding activity, although full nonspecific DNA binding affinity is retained. Surprisingly, the majority of tumorigenic mutations that destabilize DBD do not appreciably destabilize apoDBD. The R175H mutation instead substantially accelerates the rate of Zn(2+) loss. A considerable fraction of cellular p53 may therefore exist in the folded zinc-free form, especially when tumorigenic mutations are present. ApoDBD appears to promote aggregation of zinc-bound DBD via a nucleation-growth process. These data provide an explanation for the dominant negative phenotype exhibited by many mutations. Through a combination of induced p53 aggregation and diminished site-specific DNA binding activity, Zn(2+) loss may represent a significant inactivation pathway for p53 in the cell.     

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.