Structural basis of DNA recognition by p53 tetramers

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.     

Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion

The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.     

p53 DNA binding can be modulated by factors that alter the conformational equilibrium.

The p53 tumor suppressor protein is a dimer of dimers that binds its consensus DNA sequence (containing two half-sites) as a pair of clamps. We show here that after one wild-type dimer of a tetramer binds to a half-site on the DNA, the other (unbound) dimer can be in either the wild-type or the mutant conformation. An equilibrium state between these two conformations exists and can be modulated by two types of regulators. One type modifies p53 biochemically and determines the intrinsic balance of the equilibrium. The other type of regulator binds directly to one or both dimers in a p53 tetramer, trapping each dimer in one or the other conformation. In the wild-type conformation, the second dimer can bind to the second DNA half-site, resulting in drastically enhanced stability of the p53-DNA complex. Importantly, a genotypically mutant p53 can also be in equilibrium with the wild-type conformation, and when trapped in this conformation can bind DNA.     

p53 contains large unstructured regions in its native state

The human tumor suppressor protein p53 is understood only to some extent on a structural level. We performed a comprehensive biochemical and biophysical structure-function analysis of p53 full-length protein and p53 fragments. The analysis showed that p53 and the fragments investigated form stable functional units. Full-length p53 and the tetrameric fragment N93p53 (residues 93-393) are, however, destabilized significantly compared to the monomeric core domain (residues 94-312) and the monomeric fragment p53C312 (residues 1-312). At the physiological temperature of 37 degrees C and in the absence of modifications or stabilizing partners, wild-type p53 is more than 50% unfolded correlating with a 75% loss in DNA-binding activity. Furthermore the analysis of CD spectra revealed that full-length p53 contains large unstructured regions in its N and C-terminal parts. Our results indicate that full-length p53 is a modular protein consisting of defined structured and unstructured regions. We propose that p53 belongs to the growing family of loosely folded or partially unstructured native proteins. The lack of a rigid structure combined with the low overall stability may allow the physiological interaction of p53 with a multitude of partner proteins and the regulation of its turnover.     

Mechanism of folding and assembly of a small tetrameric protein domain from tumor suppressor p53

We have analyzed the folding pathway of the tetramerization domain of the tumor suppressor protein p53. Structures of transition states were determined from phi-values for 25 mutations, including leucine to norvaline, and the analysis encompassed nearly every residue in the domain. Denatured monomers fold and dimerize, through a transition state with little native structure, to form a transient, highly structured dimeric intermediate. The intermediate dimerizes, through a native-like transition state with the primary dimers fully folded but with interdimer interactions only partially formed, to form the native tetramer as a 'dimer of dimers'.     

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

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.     

Structure and Function of p53-DNA Complexes with Inactivation and Rescue Mutations: A Molecular Dynamics Simulation Study

The tumor suppressor protein p53 can lose its function upon DNA-contact mutations (R273C and R273H) in the core DNA-binding domain. The activity can be restored by second-site suppressor or rescue mutations (R273C_T284R, R273H_T284R, and R273H_S240R). In this paper, we elucidate the structural and functional consequence of p53 proteins upon DNA-contact mutations and rescue mutations and the underlying mechanisms at the atomic level by means of molecular dynamics simulations. Furthermore, we also apply the docking approach to investigate the binding phenomena between the p53 protein and DNA upon DNA-contact mutations and rescue mutations. This study clearly illustrates that, due to DNA-contact mutants, the p53 structure loses its stability and becomes more rigid than the native protein. This structural loss might affect the p53-DNA interaction and leads to inhibition of the cancer suppression. Rescue mutants (R273C_T284R, R273H_T284R and R273H_S240R) can restore the functional activity of the p53 protein upon DNA-contact mutations and show a good interaction between the p53 protein and a DNA molecule, which may lead to reactivate the cancer suppression function. Understanding the effects of p53 cancer and rescue mutations at the molecular level will be helpful for designing drugs for p53 associated cancer diseases. These drugs should be designed so that they can help to inhibit the abnormal function of the p53 protein and to reactivate the p53 function (cell apoptosis) to treat human cancer.     

Crystal structure of a superstable mutant of human p53 core domain. Insights into the mechanism of rescuing oncogenic mutations

Most of the cancer-associated mutations in the tumor suppressor p53 map to its DNA-binding core domain. Many of them inactivate p53 by decreasing its thermodynamic stability. We have previously designed the superstable quadruple mutant M133L/V203A/N239Y/N268D containing the second-site suppressor mutations N239Y and N268D, which specifically restore activity and stability in several oncogenic mutants. Here we present the x-ray structure of this quadruple mutant at 1.9 A resolution, which was solved in a new crystal form in the absence of DNA. This structure reveals that the four point mutations cause only small local structural changes, whereas the overall structure of the central beta-sandwich and the DNA-binding surface is conserved. The suppressor mutation N268D results in an altered hydrogen bond pattern connecting strands S1 and S10, thus bridging the two sheets of the beta-sandwich scaffold in an energetically more favorable way. The second suppressor mutation N239Y, which is located in close proximity to the DNA-binding surface in loop L3, seems to reduce the plasticity of the structure in large parts of loop L3 as indicated by decreased crystallographic temperature factors. The same is observed for residues in the vicinity of the N268D substitution. This increase in rigidity provides the structural basis for the increase in thermostability and an understanding how N268D and N239Y rescue some of the common cancer mutants.     

Probing phenylalanine environments in oligomeric structures with pentafluorophenylalanine and cyclohexylalanine

Stabilization of protein structures and protein-protein interactions are critical in the engineering of industrially useful enzymes and in the design of pharmaceutically valuable ligands. Hydrophobic interactions involving phenylalanine residues play crucial roles in protein stability and protein-protein/peptide interactions. To establish an effective method to explore the hydrophobic environments of phenylalanine residues, we present a strategy that uses pentafluorophenylalanine (F5Phe) and cyclohexylalanine (Cha). In this study, substitution of F5Phe or Cha for three Phe residues at positions 328, 338, and 341 in the tetramerization domain of the tumor suppressor protein p53 was performed. These residues are located at the interfaces of p53-p53 interactions and are important in the stabilization of the tetrameric structure. The stability of the p53 tetrameric structure did not change significantly when F5Phe-containing peptides at positions Phe328 or Phe338 were used. In contrast, the substitution of Cha for Phe341 in the hydrophobic core enhanced the stability of the tetrameric structure with a T(m) value of 100 degrees C. Phe328 and Phe338 interact with each other through pi-interactions, whereas Phe341 is buried in the surrounding alkyl side-chains of the hydrophobic core of the p53 tetramerization domain. Furthermore, high pressure-assisted denaturation analysis indicated improvement in the occupancy of the hydrophobic core. Considerable stabilization of the p53 tetramer was achieved by filling the identified cavity in the hydrophobic core of the p53 tetramer. The results indicate the status of the Phe residues, indicating that the "pair substitution" of Cha and F5Phe is highly suitable for probing the environments of Phe residues.     

Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e. the apo-form) has been reported. Here, we describe the solution structure and dynamics of apo-MDM2N and thus reveal the nature of the conformational changes in MDM2N that accompany binding of p53. The new structure suggests that p53 effects displacement of an N-terminal segment of apo-MDM2N that occludes access to the shallow end of the p53-binding cleft. MDM2N must also undergo an expansion upon binding, achieved through a rearrangement of its two pseudosymetrically related sub-domains resulting in outward displacements of the secondary structural elements that comprise the walls and floor of the p53-binding cleft. MDM2N becomes more rigid and stable upon binding p53. Conformational plasticity of the binding cleft of apo-MDM2N could allow the parent protein to bind specifically to several different partners, although, to date, all the known liganded structures of MDM2N are highly similar to one another. The results indicate that the more open conformation of the binding cleft of MDM2N observed in structures of complexes with small molecules and peptides is a more suitable one for ligand discovery and optimisation.     

The Oligomerization Landscape of Histones

In eukaryotes, DNA is packaged within nucleosomes. The DNA of each nucleosome is typically centered around an octameric histone protein core: one central tetramer plus two separate dimers. Studying the assembly mechanisms of histones is essential for understanding the dynamics of entire nucleosomes and higher-order DNA packaging. Here, we investigate canonical histone assembly and that of the centromere-specific histone variant, centromere protein A (CENP-A), using molecular dynamics simulations. We quantitatively characterize their thermodynamical and dynamical features, showing that two H3/H4 dimers form a structurally floppy, weakly bound complex, the latter exhibiting large instability around the central interface manifested via a swiveling motion of two halves. This finding is consistent with the recently observed DNA handedness flipping of the tetrasome. In contrast, the variant CENP-A encodes distinctive stability to its tetramer with a rigid but twisted interface compared to the crystal structure, implying diverse structural possibilities of the histone variant. Interestingly, the observed tetramer dynamics alter significantly and appear to reach a new balance when H2A/H2B dimers are present. Furthermore, we found that the preferred structure for the (CENP-A/H4)₂ tetramer is incongruent with the octameric structure, explaining many of the unusual dynamical behaviors of the CENP-A nucleosome. In all, these data reveal key mechanistic insights and structural details for the assembly of canonical and variant histone tetramers and octamers, providing theoretical quantifications and physical interpretations for longstanding and recent experimental observations. Based on these findings, we propose different chaperone-assisted binding and nucleosome assembly mechanisms for the canonical and CENP-A histone oligomers.     

How p53 binds DNA as a tetramer.

The p53 tumor suppressor protein is a tetramer that binds sequence-specifically to a DNA consensus sequence consisting of two consecutive half-sites, with each half-site being formed by two head-to-head quarter-sites (--><-- --><--). Each p53 subunit binds to one quarter-site, resulting in all four DNA quarter-sites being occupied by one p53 tetramer. The tetramerization domain forms a symmetric dimer of dimers, and two contrasting models have the two DNA-binding domains of each dimer bound to either consecutive or alternating quarter-sites. We show here that the two monomers within a dimer bind to a half-site (two consecutive quarter-sites), but not to separated (alternating) quarter-sites. Tetramers bind similarly, with the two dimers within each tetramer binding to pairs of half-sites. Although one dimer within the tetramer is sufficient for binding to one half-site in DNA, concurrent interaction of the second dimer with a second half-site in DNA drastically enhances binding affinity (at least 50-fold). This cooperative dimer-dimer interaction occurs independently of tetramerization and is a primary mechanism responsible for the stabilization of p53 DNA binding. Based on these findings, we present a model of p53 binding to the consensus sequence, with the tetramer binding DNA as a pair of clamps.     

A stable human p53 heterotetramer based on constructive charge interactions within the tetramerization domain

The human p53 tetramerization domain (called p53tet; residues 325-355) spontaneously forms a dimer of dimers in solution. Hydrophobic interactions play a major role in stabilizing the p53 tetramer. However, the distinctive arrangement of charged residues at the dimer-dimer interface suggests that they also contribute to tetramer stability. Charge-reversal mutations at positions 343, 346, and 351 within the dimer-dimer interface were thus introduced into p53tet constructs and shown to result in the selective formation of a stable heterotetramer composed of homodimers. More precisely, mutants p53tet-E343K/E346K and p53tet-K351E preferentially associated with each other, but not with wild-type p53tet, to form a heterodimeric tetramer with enhanced thermal stability relative to either of the two components in isolation. The p53tet-E343K/E346K mutant alone assembled into a weakly stable tetramer in solution, whereas p53tet-K351E existed only as a dimer. Moreover, these mutants did not form heterocomplexes with wild-type p53tet, illustrating the specificity of the ionic interactions that form the novel heterotetramer. This study demonstrates the dramatic importance of ionic interactions in altering the stability of the p53 tetramer and in selectively creating heterotetramers of this protein scaffold.     

Expanding the prion concept to cancer biology: dominant-negative effect of aggregates of mutant p53 tumour suppressor

p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases.     

p53 C-terminal interaction with DNA ends and gaps has opposing effect on specific DNA binding by the core

In addition to binding DNA in a sequence-specific manner, the p53 tumour suppressor protein can interact with damaged DNA. In order to understand which structural features in DNA the C-teminal domain recognises we have studied the interaction of p53 protein with different types of DNA oligonucleotides imitating damaged DNA. Here we show that one unpaired nucleotide within double-stranded (ds)DNA is sufficient for recognition by the p53 C-terminus, either as a protruding end or as an internal gap in dsDNA. C-terminal interaction with DNA ends facilitated core domain binding to DNA, whereas interaction with gaps prevented core domain–DNA complexing, implying that p53 might adopt distinct conformations upon binding to different DNA lesions. These observations suggest that both single-strand and double-strand breaks can serve as a target for p53 C-terminal recognition in vivo and indicate that p53 might recruit different repair factors to the sites of damaged DNA depending on the type of lesion.