Phosphodiesterases (PDEs) are a superfamily of enzymes that degrade the intracellular second messengers cyclic AMP and cyclic GMP. As essential regulators of cyclic nucleotide signalling with diverse physiological functions, PDEs are drug targets for the treatment of various diseases, including heart failure, depression, asthma, inflammation and erectile dysfunction. Of the 12 PDE gene families, cGMP-specific PDE5 carries out the principal cGMP-hydrolysing activity in human corpus cavernosum tissue. It is well known as the target of sildenafil citrate (Viagra) and other similar drugs for the treatment of erectile dysfunction. Despite the pressing need to develop selective PDE inhibitors as therapeutic drugs, only the cAMP-specific PDE4 structures are currently available. Here we present the three-dimensional structures of the catalytic domain (residues 537-860) of human PDE5 complexed with the three drug molecules sildenafil, tadalafil (Cialis) and vardenafil (Levitra). These structures will provide opportunities to design potent and selective PDE inhibitors with improved pharmacological profiles.

The c-jun N-terminal kinase (JNK) signaling pathway is regulated by JNK-interacting protein-1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the Nand C-terminal domains of JNK1 and distorts the ATPbinding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1-bound JNK1 complexed with SP600125, an ATP-competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.

In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid-starved Mesh1 null mutant has highly downregulated DNA and protein synthesis-related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.

The outer membrane protein A (OmpA) plays important roles in anchoring of the outer membrane to the bacterial cell wall. The C-terminal periplasmic domain of OmpA (OmpA-like domain) associates with the peptidoglycan (PGN) layer noncovalently. However, there is a paucity of information on the structural aspects of the mechanism of PGN recognition by OmpA-like domains. To elucidate this molecular recognition process, we solved the high-resolution crystal structure of an OmpA-like domain from Acinetobacter baumannii bound to diaminopimelate (DAP), a unique bacterial amino acid from the PGN. The structure clearly illustrates that two absolutely conserved Asp271 and Arg286 residues are the key to the binding to DAP of PGN. Identification of DAP as the central anchoring site of PGN to OmpA is further supported by isothermal titration calorimetry and a pulldown assay with PGN. An NMR-based computational model for complexation between the PGN and OmpA emerged, and this model is validated by determining the crystal structure in complex with a synthetic PGN fragment. These structural data provide a detailed glimpse of how the anchoring of OmpA to the cell wall of gram-negative bacteria takes place in a DAP-dependent manner.

Phosphodiesterases (PDEs) are essential regulators of cyclic nucleotide signaling with diverse physiological functions. Because of their great market potential and therapeutic importance, PDE inhibitors became recognized as important therapeutic agents in the treatment of various diseases. Currently, there are seven PDE inhibitors on the market, and the pharmacological and safety evaluations of many drug candidates are in progress. Three-dimensional (3D) structures of catalytic domains of PDE 1, -3, -4, -5 and -9 in the presence of their inhibitors are now available, and can be utilized for rational drug design. Recent advances in molecular pharmacology of PDE isoenzymes resulted in identification of new potential applications of PDE inhibitors in various therapeutic areas, including dementia, depression and schizophrenia. This review will describe the latest advances in PDE research on 3D structural studies, the potential of therapeutic applications and the development of drug candidates.

The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alpha CTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop. Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

In eukaryotic cells, apoptosis and cell cycle arrest by the Ras 3 RASSF 3 MST pathway are controlled by the interaction of SARAH (for Salvador/Rassf/Hippo) domains in the C-terminal part of tumor suppressor proteins. The Mst1 SARAH domain interacts with its homologous domain of Rassf1 and Rassf5 (also known as Nore1) by forming a heterodimer that mediates the apoptosis process. Here, we describe the homodimeric structure of the human Mst1 SARAH domain and its heterotypic interaction with the Rassf5 and Salvador (Sav) SARAH domain. The Mst1 SARAH structure forms a homodimer containing two helices per monomer. An antiparallel arrangement of the long ␣-helices (h2/h2 ) provides an elongated binding interface between the two monomers, and the short 3 10 helices (h1/h1 ) are folded toward that of the other monomer. Chemical shift perturbation experiments identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. The linker region between the kinase and the SARAH domain is shown to be disordered in the free protein. These results imply a novel mode of interaction with RASSF family proteins and provide insight into the mechanism of apoptosis control by the SARAH domain.tumor suppressor ͉ cell cycle arrest ͉ Hippo ͉ Salvador R ecent work in cellular homeostasis has uncovered a pathway mediated by the MST (mammalian sterile 20-like kinase) family, the human ortholog for Hippo (Hpo), which promotes apoptosis and restricts cell proliferation in conjunction with RASSF family tumor suppressors and/or the scaffold protein Salvador (Sav) (1-6). This pathway is characterized by a unique interaction motif called SARAH (for Sav/Rassf/Hpo), which connects the proteins involved in this pathway (7).Mammalian sterile 20-like kinase 1 (Mst1, also called STK4) is a member of a family of serine/threonine kinases that show similarity to Ste20, an upstream activator of the MAPK pathway in budding yeast (8,9). Mst1 is cleaved by caspase 3, which is triggered either by the activation of death receptors, such as Fas and the TNF-␣ receptor, or by exposure of the cells to inducers of apoptosis, such as staurosporine or ceramide (10-13). Whereas intact Mst1 is localized predominantly in the cytoplasm, the catalytic fragment of Mst1 generated by caspase-mediated cleavage translocates to the nucleus and phosphorylates histone H2B at Ser-14, resulting in chromatin condensation, DNA fragmentation, and, ultimately, cell death by apoptosis (14,15).A Drosophila homolog of Mst1/2, Hippo (Hpo), together with Salvador (Sav) and Warts (Wts), promotes both proper exit from the cell cycle and apoptosis during development (1-3). Mst1 and Mst2 have also been shown to associate with members of the RASSF family of tumor suppressors, such as Rassf1 and Rassf5 (also known as Nore1), all of which contain a conserved Rasassociation (RA) domain, with both of the MST and RASSF proteins colocalizing to microtubules throughout the cell cycle (4-6). Whereas purified recombinant Rassf1A inhibited the kinase activity of ...

Absent, small, or homeotic disc1 (Ash1) is a trithorax group histone methyltransferase that is involved in gene activation. Although there are many known histone methyltransferases, their regulatory mechanisms are poorly understood. Here, we present the crystal structure of the human ASH1L catalytic domain, showing its substrate binding pocket blocked by a loop from the post-SET domain. In this configuration, the loop limits substrate access to the active site. Mutagenesis of the loop stimulates ASH1L histone methyltransferase activity, suggesting that ASH1L activity may be regulated through the loop from the post-SET domain. In addition, we show that human ASH1L specifically methylates histone H3 Lys-36. Our data implicate that there may be a regulatory mechanism of ASH1L histone methyltransferases.Nucleosomes, the fundamental unit of the highly ordered chromatin structure, are composed of DNA wrapped around histone octamers. Histones have N-terminal tails that are exposed on the outside of nucleosomes. These tails are subjected to several post-translational modifications, including acetylation, phosphorylation, ubiquitination, sumoylation, and methylation (1).The site-specific methylation of histone lysine residues is important for the epigenetic control of gene expression. These marks serve to regulate epigenetically the organization of chromatin structure and to recruit other chromatin modifiers (2, 3). Methylation can occur at multiple lysine residues, including lysines 4, 9, 27, 36, and 79 of histone H3 and lysine 20 of histone H4. Absent, small, or homeotic disc1 (Ash1) is a member of the trithorax group proteins, which are essential for epigenetic gene activation (4,5). Previous studies have shown that Drosophila Ash1 activates homeotic gene ultrabithorax expression in imaginal discs of the third leg (6) and interacts with trithorax to regulate and maintain ultrabithorax expression (7). It has also been reported that the mammalian homolog of Ash1, ASH1L, is a histone methyltransferase (HMTase) 2 that is associated with transcribed regions of active genes (8, 9). ASH1L has several domains, including an associated with SET domain (AWS), a SET domain, a post-SET domain, a bromodomain, a bromoadjacent homology domain (BAH), and a plant homeodomain finger (SMART database (10)). The SET domain in HMTases is responsible for catalyzing the formation of monomethylated, dimethylated, and trimethylated lysine, establishing an additional complex system with respect to methylated lysine recognition in signaling (11). Several crystal structures of SET domain proteins have been solved, and they revealed that the SET domain forms a knot-like structure that constitutes the active site of HMTases (12). Notably, ASH1L contains a SET domain in the middle of the protein, whereas other proteins possess a SET domain at the C terminus. Drosophila Ash1 has been previously shown to affect H3K4 methylation levels genetically and to methylate H3K4, H3K9, and H4K20 in in vitro assays (8, 13). The Tanaka group (14) has found H3K36 ...