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.     

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.     

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.     

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.     

Secondary and tertiary structural changes in gamma delta resolvase: comparison of the wild-type enzyme, the I110R mutant, and the C-terminal DNA binding domain in solution.

gamma delta Resolvase is a site-specific DNA recombinase (M(r) 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catalytic core of approximately 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase. No increase in the helicity of the linker region of I110R resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.     

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.     

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.     

Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman,Atomic Force and Surface Plasmon Resonance spectroscopies

BackgroundMutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in > 50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding.MethodsMolecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28.ResultsWe show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge.ConclusionsThese results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function.General significanceRaman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53.     

Regulation of p53 nuclear export through sequential changes in conformation and ubiquitination

Wild-type p53 is a conformationally labile protein that undergoes nuclear-cytoplasmic shuttling. MDM2-mediated ubiquitination promotes p53 nuclear export by exposing or activating a nuclear export signal (NES) in the C terminus of p53. We observed that cancer-derived p53s with a mutant (primary antibody 1620-/pAb240+) conformation localized in the cytoplasm to a greater extent and displayed increased susceptibility to ubiquitination than p53s with a more wild-type (primary antibody 1620+/pAb240-) conformation. The cytoplasmic localization of mutant p53s required the C-terminal NES and an intact ubiquitination pathway. Mutant p53 ubiquitination occurred at lysines in both the DNA-binding domain (DBD) and C terminus. Interestingly, Lys to Arg mutations that inhibited ubiquitination restored nuclear localization to mutant p53 but had no apparent effect on p53 conformation. Further studies revealed that wild-type p53, like mutant p53, is ubiquitinated by MDM2 in both the DBD and C terminus and that ubiquitination in both regions contributes to its nuclear export. MDM2 binding can induce a conformational change in wild-type p53, but this conformational change is insufficient to promote p53 nuclear export in the absence of MDM2 ubiquitination activity. Taken together, these results support a stepwise model for mutant and wild-type p53 nuclear export. In this model, the conformational change induced by either the cancer-derived mutation or MDM2 binding precedes p53 ubiquitination. The addition of ubiquitin to DBD and C-terminal lysines then promotes nuclear export via the C-terminal NES.     

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.     

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.     

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.     

Property of the M-DNA probed by a minor groove binding dye 4',6-diamidino-2-phenylindole

Spectral properties including circular and linear dichroism (CD and LD) of M-DNA, a molecular electric wire, formed at a high Zn(2+) concentration have been studied using a minor groove binding drug 4',6-diamidino-2-phenylindole (DAPI) as a probe. As the Zn(2+) concentration increased, the magnitude of LD in the DNA absorption region decreased at pH 8.5, implying the aggregation of DNA, which is in contrast with the retained LD magnitude at pH 7.0. As the M-DNA formed, change in the secondary structure of DNA was observed by CD spectrum, which resembles that of the C-form DNA, although overall structure seemed to remain as a right handed double helix. The DAPI-DNA complex in the presence of high concentration of Zn(2+) ions at pH 7.0 exhibited the similar CD spectrum with that in the absence of Zn(2+) ion, consisting of type I, II and III. In contrast, at pH 8.5 at a high Zn(2+) concentration in which DNA is in its M-form, DNA bound DAPI produced only the type III CD, suggesting that DAPI binds at the surface of M-DNA: the presence of Zn(2+) ions prevents the minor groove binding of DAPI.     

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.