The single-stranded-DNA-binding proteins (SSBs) are essential for DNA function in prokaryotic and eukaryotic cells, mitochondria, phages and viruses. The structures of four SSBs have been solved, but the molecular details of the interaction of SSBs with DNA remain speculative. We report here the crystal structure at 2.4 A resolution of the single-stranded-DNA-binding domain of human replication protein A (RPA) bound to DNA. Replication protein A is a heterotrimeric SSB that is highly conserved in eukaryotes. The largest subunit, RPA70, binds to single-stranded (ss)DNA and mediates interactions with many cellular and viral proteins. The DNA-binding domain, which lies in the middle of RPA70, comprises two structurally homologous subdomains oriented in tandem. The ssDNA lies in a channel that extends from one subdomain to the other. The structure of each RPA70 subdomain is similar to those of the bacteriophage SSBs, indicating that the mechanism of ssDNA-binding is conserved.

The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8±10 nucleotides (nt)] and stable (~30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 A Ê structure of the RPA trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70 (DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)-fold and¯anked by a helix at the C-terminus. Trimerization is mediated by three C-terminal helices arranged in parallel. The OB-fold of DBD-C possesses unique structural features; embedded zinc ribbon and helix±turn±helix motifs. Using time-resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8±10 to 30 nt mode is mediated by DNA binding with the trimerization core.

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 magnesium ion, Mg 2+ , is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg 2+ uptake system of most prokaryotes 1-3 and a functional homologue of the eukaryotic mitochondrial magnesium transporter 4 . Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 Å and 1.85Å resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg 2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intra-cellular concentration of Mg 2+ . The CorA magnesium transporter is a homopentamer with fivefold symmetry about a central pore and can be divided into three parts (Fig. 1). A carboxy-terminal transmembrane domain comprises two transmembrane helices from each monomer (Fig. 2). The middle portion resembles a funnel, narrow at the entrance ( 5 Å) and wide at the mouth ( 20Å), that is formed largely by a long -helix extension of the inner transmembrane helix. Finally, a large cytoplasmic domain lies exterior to the funnel.The cytoplasmic domain of CorA is a seven-stranded parallel/antiparallel -sheet ( 2 1 3 7 6 5 4 ) sandwiched between two sets of -helices ( 1, 2, 3) and ( 4, 5, 6) ( Fig. 1). The domain fold is unlike all other known structures of ion channels or transporters and constitutes a new protein fold (see Supplementary Information). This domain, solved in its soluble form at 1.85 Å resolution ( Supplementary Fig. S1), is linked to the transmembrane helices by the long 7 helix (residues 251-312), termed the stalk helix. The stalk helix kinks as it enters the membrane, extends through the membrane, forms the first transmembrane helix (TM1; residues 293-312) and harbours the 'YGMNF' signature sequence of CorA (residues 311-315) 5,6 ( Fig. 2 and Supplementary Fig. S2). The five TM1 helices (residues 293-312) form the pore. After a short extracellular seven-amino-acid loop, the TM2 helix (residues 326-345) returns back to the cytoplasm and ends in a highly conserved C-terminal KKKKWL motif (Fig. 3). In the current structure, neither the extracellular loop nor the final two amino acids could be resolved.The cytoplasmic domain shows the lowest sequence conservati...

Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair. We have shown that a globular domain at the C terminus of subunit RPA32 contains a specific surface that interacts in a similar manner with the DNA repair enzyme UNG2 and repair factors XPA and RAD52, each of which functions in a different repair pathway. NMR structures of the RPA32 domain, free and in complex with the minimal interaction domain of UNG2, were determined, defining a common structural basis for linking RPA to the nucleotide excision, base excision, and recombinational pathways of repairing damaged DNA. Our findings support a hand-off model for the assembly and coordination of different components of the DNA repair machinery.

The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique “chemical fingerprints” for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural “priming” of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone.

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.The level of histone acetylation is regulated by the action of two classes of enzymes, histone acetyltransferases and histone deacetylases (HDACs).3 Histone acetyltransferases and HDACs are found in large multiprotein complexes, and recruitment of histone acetylase or deacetylase complexes by coactivators or corepressors is thought to cause a local change in the chromatin structure, resulting in either activation or repression of gene transcription (1). Humans have 18 HDACs and, based on their sequence similarity to yeast factors, they are grouped into four classes (class I-IV). Class II HDACs are homologous to yeast histone deacetylase HDA1 and have been implicated as global regulators of gene expression during cell differentiation and development (2). In humans, class II HDACs are subdivided into classes IIa (HDAC4, HDAC5, HDAC7, and HDAC9) and IIb (HDAC6 and HDAC10). Class IIa HDACs contain two functionally important regions, a highly conserved C-terminal catalytic domain and an N-terminal extension that has no similarity with other proteins, mediates the signal-dependent shuttling between the nucleus and the cytoplasm, and harbors binding sites for transcriptional regulators (2, 3). Class IIa HDACs interact with corepressors such as N-CoR (nuclear receptor corepressor) and the MEF2 (myocyte enhancer factor 2) family of transcription factors that is not only important for controlling gene expression in normal cellular programs like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation but has also been linked to cardiac hypertrophy, asthma, atherosclerosis, hypertension, and other pathological conditions (3-5). To date all four class IIa HDACs have been knocked out in mice, and the resulting abnormal phenotypes have been extensively characterized (6 -9). HDAC7 for example, plays an important role in cardiova...

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP, the latent origin of DNA replication in Epstein-Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1-origin interaction.