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 influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins. S tructural proteomics, which aims to determine the threedimensional (3D) structures of all proteins, has become a major initiative within the biomedical community (see ref. 1 and other articles in the same issue). The large number of protein structures expected from these projects will yield valuable clues to the rules for predicting protein folding and understanding biochemical function. In these early stages of the structural proteomics effort, one of the main goals is to identify the best technologies and the most efficient processes to convert gene sequence into 3D structural information. One of the decisions will be to determine the optimal use of x-ray crystallography and NMR spectroscopy, which are the two techniques that will provide the majority of experimental data for these initiatives.X-ray crystallography currently is perceived as the potential workhorse for structural proteomics, because if provided with a well diffracting crystal it is possible to determine a 3D structure in hours. However, the throughput of structure determination using x-ray crystallography remains unclear, because the ratedetermining step continues to be the production of well diffracting crystals, a process that is unpredictable and can take between hours and months.NMR structure determination is limited currently by size constraints and lengthy data collection and analysis times (often months), and the method is best applied to proteins smaller than 250 amino acids. On the other hand, NMR experiments do not require crystals, and samples appropriate for structure determination can be identified within minutes of the protein being purified. In summary, x-ray crystallography and NMR spectroscopy seem to have complementary deficiencies, and the relative success of these methods in structural proteomics remains to be determined.We have shown previously that NMR spectroscopy can play a significant role in structural proteomics even with its current limitations (2). The initial pilot project, based on a limited number of proteins from the thermophilic archaebacterium Methanobacterium thermoautotrophicum (Mth) suggested that smaller proteins may be more amenable to structure analysis, because in this genome a higher proportion of smaller proteins were soluble compar...

SUMMARY The phosphatase calcineurin, target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIXIT and LxVP sites. While many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATc1, and interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

Lung cancer (LC) is responsible for most cancer deaths. One of the main factors contributing to the lethality of this disease is the fact that a large proportion of patients are diagnosed at advanced stages when a clinical intervention is unlikely to succeed. In this study, we evaluated the potential of metabolomics by 1H-NMR to facilitate the identification of accurate and reliable biomarkers to support the early diagnosis and prognosis of non-small cell lung cancer (NSCLC).We found that the metabolic profile of NSCLC patients, compared with healthy individuals, is characterized by statistically significant changes in the concentration of 18 metabolites representing different amino acids, organic acids and alcohols, as well as different lipids and molecules involved in lipid metabolism. Furthermore, the analysis of the differences between the metabolic profiles of NSCLC patients at different stages of the disease revealed the existence of 17 metabolites involved in metabolic changes associated with disease progression.Our results underscore the potential of metabolomics profiling to uncover pathophysiological mechanisms that could be useful to objectively discriminate NSCLC patients from healthy individuals, as well as between different stages of the disease.