Structure of p73 DNA-binding domain tetramer modulates p73 transactivation

Abstract

The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation.

The p73 transcription factor that belongs to the p53 protein family and participates in pheromonal sensory, chromosome stability, neurogenesis, inflammation, and osteoblastic differentiation pathways (1, 2). In contrast to p53, p73 is mutated in less than 0.5% of human tumors (3); however, it also participates in p53-dependent and independent pathways, showing oncogenic and tumor suppressor functions (4, 5). These dual opposite activities are due to the presence of two promoters which results in the expression of two main isoforms, TAp73 and ΔNp73 (6).

How the members of the p53 protein family trigger different cellular responses still remains an open question. Overall, p73 and p63 can bind to the same p53 response elements (REs), but the activated pathways are different (7, 8). There is some redundancy in the activation of stress pathways by the three members of the p53 protein family, but, at the same time, over 100 genes regulated by p73 and p63 are not activated by p53 (9, 10). Like p53, p73 also binds to a 20-bp RE, comprising two half-site decamers in direct orientation that follow a 5′-Pur1-Pur2-Pur3-Cyt4-Ade5/Thy5-Ade6/Thy6-Gua7-Pyr8-Pyr9-Pyr10-3′ consensus sequence (10, 11). Half of the known p53 REs do not have any insertion between the two half-sites and spacers larger than 3 bp are rare, particularly among sites that are transcriptionally activated (12–14). In the case of p53 repressed genes, the cis-element code is poorly defined, but based on a limited number of examples, spacer length appears to be more uniformly distributed and targets have no preference for 0-bp spacers (12).

Human p73α is a 636 amino acid protein with a tripartite domain organization similar to its close homolog, p63, and to the shorter 393 amino acid long p53 protein. Members of the p53 family have a disordered N-terminal transactivation domain, a central immunoglobulin-like DNA-binding domain (DBD), and a C terminus that starts with a domain that promotes oligomerization. In p53, the last 30 amino acids form a regulatory domain that binds DNA nonspecifically, whereas p73 and p63 have more than 200 extra amino acids that include a protein–protein interaction sterile alpha-motif domain. The DBD is the most conserved domain with 58% sequence identity between p73 and p53 (Fig. 1A). The first structure of p53 DBD bound to DNA showed a loop-sheet-helix motif contacting the bases and the phosphate backbone of one quarter-site RE (15). More recent structures of p53 and p63 DBDs in complex with DNA have shown a dimer of DBD dimers where each monomer binds to one of the four basic 5-bp inverted repeat recognition sequences, creating dimerization and tetramerization interfaces (16–23), but no structure of p73 in complex with DNA is known.

Fig. 1.

Primary, secondary, and tertiary structure of human p73 DBD. (A) Sequence alignment of human DBDs of p53 protein family. Residues forming each secondary structure element are enclosed in boxes. Residues involved in zinc binding (blue), DNA binding (orange), dimerization (green), and tetramerization (gray) are highlighted. For the residues in the tetramerization interface of structures with 0-, 1-, and 2-bp RE spacers, the monomers contributing to the tetramerization interface are listed. (B) Protein-fold of p73 DBD. Secondary structure elements, polypeptide termini, and the zinc atom are labeled.

In spite of the structural knowledge accumulated, the molecular mechanism of differential transcriptional activation by p53-protein-family members remains largely unexplained. Spacer length between RE half-sites plays an important activator role to trigger apoptotic or nonapoptotic pathways (12, 24). We determined the crystal structures of p73 DBD tetramer in complex with different REs. For REs with different spacer lengths, we studied the structural basis of p73 DBD oligomerization and DNA binding and, using a yeast-based functional assay, we measured the spacer length effect on the transactivation levels induced by p73 and p53. Our results describe the oligomerization state and the changes in p73 DBD quaternary structure and DNA conformation as a function of RE spacer length, measure DNA-binding, and establish that transcriptional activity is affected more by spacer length in p73 than in p53.

Results

Crystal Structures of p73 DNA-Binding Domain with 0,1, 2, and 4 Base-Pair Spacers.

Cocrystallization experiments between p73 DBD and oligonucleotides carrying half-site REs were performed with a 198 amino acid protein construct from residues 115 to 312 of human full-length p73. We determined the crystal structure of p73 DBD in complex with DNA in three crystal forms. The structures were solved by molecular replacement using a p53 DBD dimer–DNA complex as a search model and final refined structures were determined (SI Text and Table S1). The crystal structures of p73 DBD, as in the case of p53 and p63 DBDs structures, show an immunoglobulin-like β-sandwich fold with two antiparallel β-sheets (Fig. 1B and Fig. S1). One β-sheet has four β-strands (S1, S3, S5, and S8) and the other has five β-strands (S4, S6, S7, S9, and S10). Three long loops emerge from the core β-sandwich fold. Loop L1 links β-strands S1 and S3 and contains two small β-strands, S2 and S2′, that pack against S10 and H2. The long loop L2, divided in L2A and L2B, has α-helix H1 and has a two amino acid insertion with respect to p53. Loop L3 extends from S8 to S9. A Zn²⁺ ion, crucial for dimerization and DNA binding, is tetrahedrally coordinated by Cys194 and His197 from α-helix H1 and Cys258 and Cys262 from loop L3. DNA is bound by a loop-sheet-helix motif formed by L1, L3, S10, and H2. The structure of the p73 DBD to 1.8-Å resolution in the absence of DNA has been deposited in the Protein Data Bank (2XWC; Alex Bullock Laboratory at Structural Genomics Consortium, Oxford University). The rmsd between the positions of the C^α of p73 DBD with and without DNA is 0.8 Å. The small differences are in the loops involved in tetramerization and DNA binding, particularly residues Gly265, Met266, and Asn267 are disordered in the absence of DNA and become ordered upon binding to DNA. Overall, the DBD of the members of the p53 protein family are structurally related.

Considering crystal packing, the three solved crystal forms contain five unique quaternary arrangements. The three oligonucleotides used in crystallization have closely related half-site consensus sequences, each with two identical inverted RE quarter-sites, plus one or two flanking nucleotides (Fig. 2). In the asymmetric unit of crystal 1 and 2 with 12 bp oligonucleotides, two unique tetramers bind to a central 20-bp RE (Fig. S2 A and B). The first tetramer is different in each crystal form: in crystal 1, tetramer formation displaces two base pairs, one at the end of each stacked oligonucleotide, resulting in a tetramer bound to a 0-bp spacer RE (Fig. 2A); in crystal 2, the tetramer displaces only one base pair at the juncture of both stacked oligonucleotides, resulting in a tetramer bound to a 1-bp spacer RE (Fig. 2B). The second tetramer in the asymmetric unit has an identical arrangement in both crystals without displacing any base pairs; both oligonucleotides stack on top of each other to form a tetramer complexed to a 2-bp spacer RE (Fig. 2C). In crystal 3, the asymmetric unit contains three identical p73 DBD dimers bound to three 14 bp oligonucleotides where dimers separated by 4 bp do not form a dimer–dimer interface (Fig. 2D and Fig. S2C). The DNA packing in the three crystals forms (Fig. S2 D and E) results in the stacking of two oligonucleotides to form a double-stranded full-length RE with continuous electron density (Fig. S3); the existence of a continuous DNA density was confirmed by analyzing the DNA conformation and carrying out extra refinement steps with the DNA ends joined to model entire REs for each tetramer in the three crystal forms (Fig. S4).

Fig. 2.

Crystal structures of p73 DBD in complex with DNA. (A) Tetramer bound to two 12 bp oligonucleotides forming an RE with 0-bp spacer with two base pairs flipped out of the DNA double helix. (B) Tetramer bound to two 12 bp oligonucleotides forming an RE with 1-bp spacer (in black) with one base pair flipped out of the DNA double helix. (C) Tetramer bound to two 12 bp oligonucleotides forming an RE with 2-bp spacer (in black). (D) Dimers bound to a 14 bp oligonucleotide with half-site RE. Two oligonucleotides stack to form a RE with a 4-bp spacer (in black) with dimers not forming a tetramer.

The p73 DBD Recognizes Different REs in a Structurally Similar Manner.

To understand how p73 DBD binds DNA, we studied its oligomerization by analytical ultracentrifugation. Sedimentation velocity experiments demonstrated that p73 DBD is a monomer in the absence of DNA and it dimerizes upon DNA binding (Fig. 3A). In the absence of DNA, the isolated p73 DBD is a monomer with a 2.1 S sedimentation coefficient. However, experiments with fluorescein-labeled oligonucleotides of different lengths containing either half- (12 or 14 bp) or full-site REs (20 or 24 bp) show that p73 DBD first binds to DNA as a dimer with a sedimentation coefficient between 3.4 and 4.2 S and, when the oligonucleotide includes a full RE, besides the dimer, a DNA-bound tetramer with a sedimentation coefficient between 6.5 and 6.8 S appears. The oligomeric forms observed in the crystal packing of p73 DBD in complex with DNA are consistent with the hydrodynamic experiments (Figs. 2 and 3A).

Fig. 3.

Oligomerization of p73 DBD and DNA binding by p73 DBD. (A) Sedimentation coefficient distribution of the oligomeric species free p73 DBD and in complex with DNA containing one (12 and 14 bp) or two half-sites (20 and 24 bp). (B) Binding affinity constants of p73 DBD for the three half-site REs used in crystallization and of p73 DBD and ΔNp73δ for full-site REs with 0-, 1-, 2-, and 4-bp spacers. (C) Crystal structure of monomer A in crystal 1 in complex with DNA showing half-site RE and the residues that contact the DNA bases and the phosphate backbone. (D) Schematic diagram of the atomic interactions between the p73 DBD and DNA.

To understand p73 DBD DNA-binding properties and the effect of space length in DNA binding, we studied the three half-site RE sequences used in the crystallization and four full-site RE sequences with 0-, 1-, 2-, and 4-bp spacers by fluorescence anisotropy (Fig. 3B and Fig. S5). A p73 DBD dimer recognizes a half-site that follows the 5′-Pur1-Pur2-Pur3-Cyt4-Ade5/Thy5-Ade6/Thy6-Gua7-Pyr8-Pyr9-Pyr10-3′ consensus rule. The p73 DBD dissociation constants obtained for p73 DBD dimer binding to the half-site RE sequences used for crystallization were similar, demonstrating that purine/purine substitutions in the first and third base pairs result in equivalent binding (Fig. 3B and Fig. S5). Importantly, the p73 oligomerization domain has an essential contribution to DNA affinity, as already observed for p53 (21, 25). These values are also comparable to the ones observed for p63 DBD (22).

The interactions between p73 DBD and DNA involve residues from a loop-sheet-helix motif (L1-S10-H2) to the DNA bases and backbone, plus interactions of loop L3 with the DNA backbone (Fig. 3C). Approaching from the DNA major groove, Arg300, Cys297, and Lys138 reach the DNA major groove to contact the DNA bases Gua4′, Cyt3′, and Gua2/Ade2, respectively (Fig. 3D). The cytosine in position four is the most conserved base of the quarter recognition site because its complementary base Gua4′ has two atoms, O6 and N7, sharing hydrogen bonds with the Arg300 guanidinium group. Purine degeneracy at positions two and three of the consensus site is due to the flexibility of Cys297 and Lys138. The sulfhydryl group from Cys297 is a hydrogen-bond acceptor to the N4 of Cyt3′ in crystal 1 and 2 and a hydrogen-bond donor to the O4 of Thy3′ in some monomers in crystal 3; although Lys138 is always hydrogen bonding to N7 of Gua2 in all the crystals forms, it is found in some monomers keeping multiple hydrogen bonds that also include the O6 of Gua3. No direct contacts are observed to the bases in positions one and five. Besides the described contacts to the DNA bases, five contacts to the DNA phosphates stabilize the complex: the amide groups of Lys138 and Ala296 and the minor-groove-approaching side chains of Ser261, Arg268, and Arg293 in strand S10. The average distance found between the C1′ atoms of the central A-T base pairs in all the crystal forms is about 10 Å, which is closer to the ideal Watson–Crick distance (Fig. S3E). The central A-T base pair was modeled as a Watson–Crick base pair because the 2.9-Å resolution of our maps did not allow us to observe the likely flip of the central Ade5 to a Hoogsteen base-pair conformation as it has been described for p53 (20).

Dependence of p73 Transactivation Activity on RE Spacer Length.

RE spacer length is an important regulatory mechanism in the p53 protein family (12). ChIP and microarray experiments have shown that p73 activates at least 85 genes, 27 of which are also activated by p53 (26). For the 85 genes activated by p73, the p53FamTaG database lists 266 p73 REs with a wide range of conservation of the consensus motif (27). Of the 50 p73 REs that have a conserved central CATG motif in both half-sites, 82% present a 0-bp spacer (Fig. 4A); in less conserved motifs, the spacer length distribution is broader (Fig. 4B).

Fig. 4.

Role of RE spacers in p73 transactivation. (A and B) Spacer length distribution of the 50 p73 REs reported in the p53FamTaG database with conserved central CATG bases and the 163 p73 REs reported with conserved central CATG bases in one half-site and another half-site with variable CNNG central bases. (C) Sequences used as enhancer of the firefly luciferase reporter gene in isogenic yeast reporter strains. The nomenclature for each RE sequence refers to the crystal form (C1, C2, or C3) and the spacer length (-SP0, -SP1, -SP2 or -SP4, in black). (D and E) Effect of sequence and spacer on p73-dependent transactivation as measured in a yeast-based functional assay. Data represent the average and standard error of four luciferase-activity assays measured for strains named in C at moderate levels of human p73β expression under the control of the constitutive ADH1 promoter and in E at high levels of expression under the inducible GAL1-10 promoter with 0.12% galactose. The average relative-light-units (RLU) were normalized by cell-number as measured with OD at 600 nm and the zero level was defined by the basal activity with an empty expression vector. (F and G) Same as D and E for p53.

To investigate the effect of RE sequence and spacer length on p73 transactivation potential, we used a yeast-based functional assay (28). In the assay, p73 REs are used as upstream enhancers of the expression of a firefly luciferase gene controlled by a minimal promoter and cloned into a constant chromosomal location in isogenic yeast strains that naturally do not contain a p73 homolog (13). In experiments with yeast cells, we previously demonstrated that human p73β protein can act as a transcription factor using constitutive and inducible promoters (29). Hence, we tested the ability of human p73β to induce the expression of the luciferase gene under the enhancer control of 10 REs with spacers from 0 to 4 bp that are variations of the three half-site sequences used for crystallization (Fig. 4C). The three 0-bp spacer consensus sequences examined showed that p73 was active as a transcription factor and revealed a different transactivation potential for each (C2-SP0 > C3-SP0≥C1-SP0) (Fig. 4 D and E). The C2 RE was the most responsive sequence of the three and it was the least affected by the insertion of a spacer between the half-sites. In the context of promoters that produce moderate (ADH1) or high (GAL1-10) levels of p73β expression, the C2 RE with 1-bp spacer (C2-SP1) retains approximately 10% of transactivation activity, whereas insertions of 2 or 4 bp (C2-SP2 and C2-SP4) reduce the transactivation response to background levels (Fig. 4 D and E). We observed a difference in the effect of spacers on the transactivation response of p73 and p53 for the sequence C2. Whereas p73 transactivation activity dropped significantly with any insertion, p53 tolerated 1-, 2-, and 4-bp spacers without a substantial drop in activity, especially at moderate levels of expression with the constitutive ADH1 promoter or at high levels with the GAL1-10 promoter (Fig. 4 F and G). For REs with C1 and C3 sequences, both p73 and p53 show similar transactivation activity without spacer, but the presence of a spacer destroys activity, except for p53 with the C3-SP2 sequence that maintain some activity. In general, the presence of spacers decreases transactivation activity and it drops more rapidly for p73 than for p53.

p73 DBD Quaternary Structure Depends on RE Spacer Length.

Besides the described protein-DNA contacts that are identical for each monomer, in order to understand the effect of RE spacer length on p73 DBD quaternary structure, one must describe the changes in dimerization, tetramerization, and DNA conformation that occur as the number of bases between the two RE halves increases (Fig. 5). Regarding protein–protein interfaces, p73 DBD tetramers have five interaction surfaces: two are monomer–monomer surface areas that stabilize the dimers (A–B and C–D) and the other three surfaces are dimer–dimer surface contacts that form the tetramer (A–D, B–C, and B–D) (Fig. S6A). As spacer length increases, the dimer–dimer distances increase and the buried surface area in the tetramer decreases (SI Text and Fig. S6B).

Fig. 5.

Protein and DNA conformational changes on p73 DBD tetramers bound to REs with different spacers. (A and B) Dimerization and tetramerization interfaces of 0 and 2 bp tetramers. In the center of the panel, we show the secondary structure elements involved in the dimerization and tetramerization interfaces of the p73 DBD tetramer. On the top and bottom panels, we show the atomic details of the amino acids forming the tetramerization and dimerization interfaces, respectively. (C) DNA conformation of the refined continuous DNA molecules. The 0- and 4-bp structures conserve a B-DNA conformation, whereas the 1- and 2-bp structures twist the spacer nucleotides to unwind the double helix and allow the tetramer to continue binding to the central CATG recognition sites. Extra crystallographic refinement cycles were carried out after joining the ends of the stacked half-site RE oligonucleotides used in crystallization.

As observed in the sedimentation velocity experiments, a dimer is the minimum oligomer required for p73 DBD to bind to DNA (Fig. 3A). In all the solved structures, the dimerization interfaces are the least affected by the insertion of spacers. Nonetheless, two distinct dimer conformations could be observed (Fig. 5 and Fig. S7). One dimer conformation is influenced by tetramerization, like all the dimers in the 0- and 1-bp structures, plus the LK dimer in the 2-bp structures. The other dimer conformation represents p73 DBD dimer conformation when tetramerization restraints are weaker or absent, such as in the second IJ dimer of the 2-bp structures and the dimers of the 4-bp structure. The dimerization interface is able to establish two different hydrogen-bond networks that appear to correspond to dimers in tetramers and dimers in the absence of tight tetramerization (Fig. 5 A and B).

Tetramerization interfaces are more sensitive than the dimerization surfaces to conformational arrangement. For every base pair inserted between RE half-sites, a dimer would be expected to rotate 36° with respect to the other dimer and the distance between dimers would increase by 3.4 Å; nonetheless, the structures here described indicate that, in the presence of 1- and 2-bp spacer insertions, the forces keeping the tetramer together differ from the ideal B-DNA conformation. The dimer–dimer distance in the structures with 0- and 1-bp spacers barely increases from 34 to 35 Å (Fig. S6B). Instead, in the 2-bp spacer structure, one of the tetramerization interfaces is weakened; consequently, the monomer to monomer distance increases to 40 Å, and, in the 4-bp spacer structure, there is no tetramerization interface due to the 52 Å that separates two dimers. Regarding the rotation angle between dimers, the 0- and 1-bp tetramers maintain a flat dimer-of-dimers structure (Fig. S8). In contrast, with a 2-bp spacer, the p73 DBD tetramer does not maintain intact the tetramerization interface because dimers move apart 6 Å and rotate 14° out-of-plane (instead of the expected 7 Å and 72° for a 2-bp insertion) (Figs. S6 and S8). In the 2-bp spacer structure, the tetramer is not flat and, whereas one of the two tetramerization interfaces has a large 405 Å² buried surface area that is similar to the one found for 0 and 1 bp tetramers, the other tetramerization interface is disrupted and has a smaller 282 Å² buried surface (Fig. S6B). The tetramerization interfaces are formed by hydrogen-bond contacts and a majority of hydrophobic interactions from residues, mainly, located in the loops of the monomers (Fig. 5 A and B). As the tetramerization surface area decreases, the number of total hydrogen bonds and hydrophobic contacts in the tetramerization interface also decreases as the spacer length increases and the residues forming the tetramerization interface change, particularly loop L2A (Fig. 5 and SI Text).

DNA Conformation upon Tetramer Binding Depends on RE Spacer Length.

Besides the described changes in the protein conformation of the p73 DBD tetramer, the conformation of the continuous DNA density that forms the full REs in the three crystal forms changes depending on the length of the RE spacer (Fig. 5C and Fig. S3). The DNA structure of the 0- and 4-bp spacer can be described as the classical B-DNA form; the only deviation is that the 4-bp spacer structure has a 3-Å slide in the middle of the spacer (Fig. S4 A and E). In comparison, the DNA structure in the 1- and 2-bp spacer structures show an unwinding of the DNA helix in the middle of the spacer (Fig. S4 B–D). A B-DNA conformation has a 36° twist at every step of the helix, but the 0- and 1-bp spacer structures show a 2° and -30° twist at the center of the RE spacer (Fig. 5C). Besides DNA unwinding, a slight bending toward the major groove in the same region allows to fit an extra base in the 1-bp spacer structure without distorting the quaternary structure of the tetramer. The DNA in the 2-bp spacer structure also bends slightly, but clearly not enough to compensate the extra 7 Å required to accommodate two extra base pairs without distorting the quaternary structure of the tetramer, thus some tetramer contacts break. The double-helix unwinding is the key DNA deformation that allows the tetramer to continue forming and binding to the two half-REs in spite of the additional base pairs.

Discussion

Transcription regulation is a fundamental process that underlies the molecular mechanisms of basic cellular functions, like cell growth, division, arrest, and death. We describe the quaternary structure changes in dimerization, tetramerization, and DNA conformation when p73 DBD is bound to REs of different spacer length and we measure DNA binding and in vivo p73 transactivation activity. The present manuscript shows that the distance between half-site REs affects the p73 DBD quaternary structure that acts as a scaffold to regulate p73 transactivation activity.

All the members of the p53 family have similar RE specificity (30). This work confirms that the p53 protein family has a conserved motif for DNA recognition (15–20, 22, 23, 31) (Figs. 1 and 3). The residues from the p73 DBD that contact the DNA bases (Lys138, Cys297, and Arg300) are conserved in p53 (Lys120, Cys277, and Arg280) and p63 (Lys149, Cys308, and Arg311) (Fig. 1A). Arg300 recognizes the conserved cytosine in the center of the half-site RE, Lys138 recognizes purines in positions 2 and 3, and Cys297 binds to the pyrimidine in position 3 (Fig. 3C). The p73 DBD recognizes the three DNA sequences that we studied in the same manner.

The conservation of a DNA recognition motif in the p53 protein family does not explain the different patterns of gene expression reported for p53 and p73 (7, 8). Although there is a general overlap of the p73 and p53 consensus binding sites identified by in vitro and in vivo studies, specific differences noted by SELEX, EMSA, gene reporter assays, ChIP cloning, and ChIP-sequencing analysis suggest a broader target specificity for p73 (10, 32, 33). Target specificity in the p53 protein family may partially be explained by the spacer length found between half-sites. For p53, transactivation activity is known to be affected by the number of nucleotides inserted between the two 10-bp half-sites of the full-RE (12–14). We determined the effect of RE spacer length was more drastic on the transactivation activity of p73β than for p53 and we also noted some sequence-dependent effect (Fig. 4 D and E and Fig. S6). These results suggest that p73 activation is even more sensitive to RE sequence than what has already been observed for p53 (34).

The p73 transcriptional activation is a multistep process involving DNA binding, dimerization, tetramerization, recruitment of transcriptional machinery, transcription initiation, and elongation. This study suggests that the mechanism of p73 transactivation is dependent on structural changes that occur in the oligomerization interfaces of p73 DBD tetramer upon binding to different REs. Although our structural results were obtained for p73 DBD and our transactivation results were obtained with full-length p73β, we looked for structure–function correlations that could provide some insight into how the quaternary structure of the p73 DBD–DNA complex promotes transcriptional activation. As our binding results with the ΔNp73δ isoform show, and has also been shown for p53 multidomain constructs, any effort to explain transactivation by only understanding DNA binding by the DBD is an oversimplification (22). Nevertheless, it is interesting to notice that p73 DBD quaternary structure changes correlate with the level of p73 transactivation ability. The p73 DBD tetramer bound to 0- or 1-bp spacer REs is a flat tetramer, and their transactivation activity is higher than the distorted tetramer bound to a 2-bp spacer RE, that has, if any, only basal transactivation activity.

Binding to DNA determines oligomerization and activation. Hydrodynamic experiments indicate that, in the absence of DNA, the purified p73 DBD is a monomer; then, as soon as DNA is present, p73 DBD first dimerizes on the DNA and, if a second half-site is available, it forms a tetramer (Fig. 3A). REs with 2-bp spacers or shorter allow the formation of tetramers, as in crystal forms 1 and 2 or in reported structures of p53 and p63 (17–23). When p73 DBD binds to DNA with spacers larger than 2 bp, it does not form tetramers and the inability to tetramerize might explain the lack of p73β transactivation observed in the yeast-based assay. Regarding the DNA conformation, some studies on p53 have shown DNA bending (17, 18, 20), whereas others have not (19, 21, 23). In the case of p73 DBDs, dimers bind to an undisturbed B-DNA half-site RE and bending in the half-sites is not observed; on the other hand, when two oligonucleotides stack to form the 20-bp RE with 1- or 2-bp spacers, the p73 DBD tetramer is still able to recognize both half-site REs and compensates the insertions by unwinding the DNA 30° and 60°, respectively (Fig. 5C and Figs. S3 and S4).

Interestingly, p53 DBD binding affinity for DNA is 20 to 100 times greater than for p73 and p63 DBDs, and only our results with the ΔNp73δ isoform approach such values (22, 35) (Fig. 3B). The difference in affinity cannot be explained by how DNA is recognized, but it may be due to the differences in the oligomerization interfaces that have less than 50% of residues conserved between p73 and p53 (Fig. 1). The dimerization and tetramerization interfaces for the p73 DBD are smaller than for the p53 DBD. The p53 DBD dimer is held by van der Waals interactions from Pro177, His178, Met243, and Gly244 and an intermolecular salt bridge between Glu180 from the L2 loop of one monomer with Arg181 from the other monomer (20). Instead, the salt bridge is missing in p73 because Leu199 substitutes the Arg181 found in p53 and the replacement of the Met243 seen in p53 for Val263 in p73 explains the smaller interaction surface (Fig. 5A).

Although we have described changes in the quaternary structure of p73 DBD upon DNA binding that correlate with the transactivation activity of the full-length protein (Fig. 5), how the described active p73 DBD tetramer conformation affects the transactivation activity of the full-length protein needs to be understood.

Methods

A detailed description of the methods is available in the SI Text. Human p73 DBD domain (residues 115–312) was expressed in Escherichia coli and purified to homogeneity. Commercial DNA was lyophilized and dissolved in water to a final concentration of approximately 7 mg mL^-1. For crystallization trails, a molar ratio of 4∶1 (protein:DNA) was used. The best crystallization conditions were 100 mM MES pH 6.0, 0.1 M ammonium acetate, and 12% (wt/vol) PEG 20000. Data were collected at beamline BL7-1 at Stanford Synchrotron Radiation Lightsource, structures were solved by molecular replacement and refined to reach low R-free values. Transactivation assays were carried out in yeast strains carrying a luciferase reporter gene. Sedimentation coefficients were measured in a Beckman Optima XL-I ultracentrifuge and DNA-binding constants were measured using 5′-fluorescein-labeled dsDNA in a Hitachi F-2000 fluorescence spectrophotometer.