One of many protein-protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1-120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37-57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA͞p53 interaction can be modulated. RPA70N forms an oligonucleotide͞oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37-57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide͞oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.DNA binding ͉ protein-protein interaction ͉ structural analysis ͉ ssDNA mimicry U pon DNA damage, the p53 tumor suppressor is activated and orchestrates a cellular response by transcriptional regulation of genes involved in cell cycle arrest and apoptosis (1, 2). p53 protein is central to an extensive network of DNA damage sensing proteinprotein and protein-nucleic acid interactions. As yet, however, details of how this network is regulated are unclear. One component of the network is replication protein A (RPA), the major single-stranded (ss) DNA-binding protein of the eukaryotic nucleus (3-5). The interaction of p53 with RPA mediates suppression of homologous recombination (6) and modulates Werner syndrome helicase activity (7). It is also linked with DNA repair and disruption of p53 and RPA complexes after DNA damage is thought to coordinate DNA repair with the p53-dependent checkpoint control (8).Because the ability of p53 to bind specific DNA target sequences via its DNA-binding core (9) (Fig. 1,) is blocked when the protein is complexed with RPA it follows that UV-mediated disruption of the complexes is predicted to favor p53 transactivation functions (10). p53-RPA complex formation is affected by the presence of various lengths of ssDNAs, because RPA, when bound to these ssDNAs, is unable to interact with p53 (10). UV radiation of cells also reduces p53-RPA complexes by a second mechanism, because hyperphosphorylated RPA does not associate with p53 (8). Thus p53-RPA interaction is subject (i) to the presence of ssDNA molecules and also (ii) to the phosphorylation status of the RPA protein.RPA is a heterotrimer (RPA70, RPA32, and RPA14; Fig. 1B) involved in many aspects of DNA metabolism such as replication, recombination, and repair (11,12). The largest subunit, RPA70, is a tandem repeat of four oligonucleotide͞oligosaccharide-binding (OB) folds (13) comprising RPA70...

The p53 tumor suppressor gene and members of the transforming growth factor-␤ (TGF-␤) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-␤ superfamily, designated placental TGF-␤ (PTGF-␤), that is upregulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-␤ is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-␤ promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-␤ promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-␤ overexpression from a recombinant adenoviral vector (AdPTGF-␤) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50 -60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-␤ overexpression was seen to induce both G 1 cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-␤ superfamily and implicate PTGF-␤ as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.

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Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human cancers. Recent identification of two human homologues of p53 has raised the prospect of functional interactions between family members via a conserved oligomerization domain. Here we report in vitro and in vivo analysis of homo-and hetero-oligomerization of p53 and its homologues, p63 and p73. The oligomerization domains of p63 and p73 can independently fold into stable homotetramers, as previously observed for p53. However, the oligomerization domain of p53 does not associate with that of either p73 or p63, even when p53 is in 15-fold excess. On the other hand, the oligomerization domains of p63 and p73 are able to weakly associate with one another in vitro. In vivo co-transfection assays of the ability of p53 and its homologues to activate reporter genes showed that a DNA-binding mutant of p53 was not able to act in a dominant negative manner over wildtype p73 or p63 but that a p73 mutant could inhibit the activity of wild-type p63. These data suggest that mutant p53 in cancer cells will not interact with endogenous or exogenous p63 or p73 via their respective oligomerization domains. It also establishes that the multiple isoforms of p63 as well as those of p73 are capable of interacting via their common oligomerization domain.Tumor suppressor p53 is a transcriptional regulator of genes involved in control of the cell cycle and/or apoptosis (reviewed in Refs. 1-3). In response to cellular stress, particularly DNA damage, p53 protein levels rise, leading to changes in expression of p53 responsive genes and subsequent cell cycle arrest and/or apoptosis. Growth arrest or cell death prevents damaged DNA from being replicated and suggests a role for p53 in maintaining the integrity of the genome (4). This DNA damage checkpoint activity is central to its role as a tumor suppressor and also of major importance in the response of many cancers to conventional therapies that trigger apoptosis by damaging DNA. Inactivation of p53 through deletion, mutation, or interaction with cellular or viral proteins is a key step in over half of all human cancers (5, 6). Reactivation of the p53 pathway leading to apoptosis in cancer cells containing inactive p53 could greatly improve current modes of treatment (7,8) and could potentially be a treatment modality in itself (9, 10).Recently, two p53 homologues have been identified that can activate some of the same target genes as p53 and can induce apoptosis. The existence of these new proteins adds new complexity to our understanding of the p53 pathway and offers new potential for its reactivation in cancer cells. Human p73 (11, 12) and p63 (13-15) share regions of homology with the activation, DNA-binding, and oligomerization domains of p53. This raises the possibility of physical, and/or genetic interactions between p53 family members (13), as is often the case within other families of transcription factors such as the homeodomain proteins (16) or the Myc superfamily (17). Both p63 a...

p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.

A mass spectrometer coupling electrospray ionization with time-of-flight mass spectrometry (ESI-TOFMS) has been used to investigate the oligomeric species of Escherichia coli citrate synthase, and to determine the effect of nicotinamide adenine dinucleotide (NADH), an allosteric inhibitor of this enzyme, on the equilibrium between the oligomeric forms. An equilibrium mixture of dimers (M = 95,770 Da) and hexamers (M = 287,310 Da) was found under the conditions used (KA = 6.9 x 10(10) M-2), and NADH was observed to bind selectively to the hexamer (KD = 1.1 microM), shifting the equilibrium to the latter form. The power of ESI-TOFMS to measure ions of very large mass-to-charge ratio (up to m/z approximately 10,000 in this case) is shown to be a valuable tool for obtaining accurate information about compositions of noncovalent complexes and equilibrium constants. The measured constants agree with those determined by conventional means.

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) has been used to study noncovalent interactions between the rrp apo-repressor (TrpR), its co-repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21-base-pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer-dsDNA complex with a 1 : 1 stoichiometry was observed. Co-repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID-MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI-MS to test the sequence-specificity of protein-DNA complexes. The interactions between the TrpR-DNA complex and 5-methyl-, L-and D-tryptophan were also investigated. Two molecules of 5-methyl-or L-tryptophan were bound with high affhity to the TrpR-DNA complex. On the other hand, D-tryptophan appeared to bind to the complex with poor specificity and poor affinity.Keywords: electrospray ionization; noncovalent; protein-DNA interaction, time-of-flight mass spectrometry, trp repressor Over the last ten years, electrospray ionization mass spectrometry (ESI-MS) has demonstrated a high potential for the characterization of biomolecules by allowing the determination of molecular masses up to about 150 kDa with an accuracy of 0.01% (Fenn et al., 1990;Mann & Wilm, 1995 such as pH, buffers, temperature, and ion kinetic energy are carefully controlled, then noncovalent interactions between molecules can also be preserved during the ionization-desorption process. This technique has been successfully used to study protein-protein (Light-Wahl et al

We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.