The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.

The BRCA1 tumor suppressor gene encodes an 1863 amino acid gene product that is implicated in many cellular pathways including transcription, cell-cycle checkpoint control, apoptosis and DNA repair. Much attention has been focused on the structural and biochemical characterization of the N-terminal RING and tandem C-terminal BRCT domains of BRCA1. Here we used NMR spectroscopy in conjunction with CD spectroscopy and limited proteolysis to investigate the biophysical properties of the approximately 1500 residue central region of BRCA1. Our results show that although there are a few small, mildly protease-resistant regions, the majority of the BRCA1 central region lacks any pre-existing independently folded globular domains. Electrophoretic mobility shift assay and intrinsic tryptophan fluorescence experiments also demonstrate that, although intrinsically disordered, polypeptides from the central region are able to mediate interactions with DNA and p53 with affinities in the low micromolar range. This supports a model in which the central region may act as a long flexible scaffold for intermolecular interactions, thereby helping to integrate multiple signals in the DNA damage response pathway.

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 RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-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 oligonucleotide/oligosaccharide-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.

Study of the hexameric and allosterically regulated citrate synthases (type II CS) provides a rare opportunity to gain not only an understanding of a novel allosteric mechanism but also insight into how such properties can evolve from an unregulated structural platform (the dimeric type I CS). To address both of these issues, we have determined the structure of the complex of NADH (a negative allosteric effector) with the F383A variant of type II Escherichia coli CS. This variant was chosen because its kinetics indicate it is primarily in the T or inactive allosteric conformation, the state that strongly binds to NADH. Our structural analyses show that the six NADH binding sites in the hexameric CS complex are located at the interfaces between dimer units such that most of each site is formed by one subunit, but a number of key residues are drawn from the adjacent dimer. This arrangement of interactions serves to explain why NADH allosteric regulation is a feature only of hexameric type II CS. Surprisingly, in both the wild-type enzyme and the NADH complex, the two subunits of each dimer within the hexameric conformation are similar but not identical in structure, and therefore, while the general characteristics of NADH binding interactions are similar in each subunit, the details of these are somewhat different between subunits. Detailed examination of the observed NADH binding sites indicates that both direct charged interactions and the overall cationic nature of the sites are likely responsible for the ability of these sites to discriminate between NADH and NAD(+). A particularly novel characteristic of the complex is the horseshoe conformation assumed by NADH, which is strikingly different from the extended conformation found in its complexes with most proteins. Sequence homology studies suggest that this approach to binding NADH may arise out of the evolutionary need to add an allosteric regulatory function to the base CS structure. Comparisons of the amino acid sequences of known type II CS enzymes, from different Gram-negative bacteria taxonomic groups, show that the NADH-binding residues identified in our structure are strongly conserved, while hexameric CS molecules that are insensitive to NADH have undergone key changes in the sequence of this part of the protein.

A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described. Virtually complete backbone (1)HN, (15)N, (13)C, and (13)C(beta) chemical shift assignments of this largely alpha-helical protein are reported. The assignment strategy follows from our previously described approach based on TROSY triple resonance 4-D NMR spectroscopy [Yang, D.; Kay, L. E. J. Am. Chem. Soc. 1999, 121, 2571-2575. Konrat, R; Yang, D; Kay, L. E. J. Biomol. NMR 1999, 15, 309-313] with a number of modifications necessitated by the large size of the protein. A protocol for refolding deuterated MSG in vitro was developed to protonate the amides deeply buried in the protein core. Of interest, during the course of the assignment, an isoaspartyl linkage in the protein sequence was unambiguously identified. Chemical shift assignments of this system are a first step in the study of how the domains of the protein change in response to ligand binding and for characterizing the dynamical properties of the enzyme that are likely important for function.

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.

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.

The p53 tumor suppressor is a transcription factor that plays a crucial role in the activation of genes in response to DNA damage. As a first step towards detailed structural studies of the molecule aimed at understanding its regulation, we have used 4D-TROSY triple resonance NMR spectroscopy to obtain nearly complete 1HN, 15N, 13Cα, 13CO and 13Cβ resonance assignments of a dimeric form of the protein comprising DNA-binding and oligomerization domains (67 kDa). A simple comparison of 4D spectra recorded on p53 molecules consisting of DNA-binding and oligomerization domains with and without the regulatory domain establishes that both constructs have essentially identical chemical shifts. Although the affinity of p53 for target DNA is decreased in constructs containing the regulatory domain, the chemical shift results reported here suggest that this decrease is not due to specific domain interactions involving the regulatory portion of the molecule, or alternatively, that such interactions require the presence of DNA.

The p53 tumor suppressor gene and members of the transforming growth factor-β (TGF-β) 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-β superfamily, designated placental TGF-β (PTGF-β), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-β 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-β 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-β promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-β overexpression from a recombinant adenoviral vector (AdPTGF-β) 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-β overexpression was seen to induce both G1cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-β superfamily and implicate PTGF-β as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.

The formation of noncovalent complexes by macro molecules is one of the most important molecular processes in biology. It is intimately involved in such recognition phenomena as enzyme-substrate interaction, receptor-ligand binding, formation of oligomeric proteins, assembly of transcription complexes, and the formation of cellular structures themselves. Established methods for studying these complexes suffer from various difficulties such as large sample requirements (X-ray crystallography), limited mass range (nuclear magnetic resonance spectroscopy), lack of specificity (ultracentrifuge), or poor mass resolution (gel electrophoresis).KeywordsBuffer ConcentrationNoncovalent ComplexMass Spectrometric MeasurementStorage RegionRadio Frequency QuadrupoleThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The p53 tumor suppressor is a transcription factor that plays a crucial role in the activation of genes in response to DNA damage. As a first step towards detailed structural studies of the molecule aimed at understanding its regulation, we have used 4D-TROSY triple resonance NMR spectroscopy to obtain nearly complete 1H(N), 15N, 13C(alpha), 13CO and 13C(beta) resonance assignments of a dimeric form of the protein comprising DNA-binding and oligomerization domains (67 kDa). A simple comparison of 4D spectra recorded on p53 molecules consisting of DNA-binding and oligomerization domains with and without the regulatory domain establishes that both constructs have essentially identical chemical shifts. Although the affinity of p53 for target DNA is decreased in constructs containing the regulatory domain, the chemical shift results reported here suggest that this decrease is not due to specific domain interactions involving the regulatory portion of the molecule, or alternatively, that such interactions require the presence of DNA.

p73 and p63 are two recently cloned genes with homology to the tumor suppressor p53, whose protein product is a key transcriptional regulator of genes involved in cell cycle arrest and apoptosis. While all three proteins share conserved transcriptional activation, DNA‐binding and oligomerization domains, p73 and p63 have an additional conserved C‐terminal region. We have determined the three‐dimensional solution structure of this conserved C‐terminal domain of human p73. The structure reveals a small five‐helix bundle with striking similarity to the SAM (sterile alpha motif) domains of two ephrin receptor tyrosine kinases. The SAM domain is a putative protein–protein interaction domain found in a variety of cytoplasmic signaling proteins and has been shown to form both homo‐ and hetero‐oligomers. However, the SAM‐like C‐terminal domains of p73 and p63 are monomeric and do not interact with one another, suggesting that this domain may interact with additional, as yet uncharacterized proteins in a signaling and/or regulatory role.