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Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of pyridine-containing compounds using the cofactor S-5'-adenosyl-l-methionine (SAM) as the methyl group donor. Through the regulation of the levels of its substrates, cofactor, and products, NNMT plays an important role in physiology and pathophysiology. Overexpression of NNMT has been implicated in various human diseases. Potent and selective small-molecule NNMT inhibitors are valuable chemical tools for testing biological and therapeutic hypotheses. However, very few NNMT inhibitors have been reported. Here, we describe the discovery of a bisubstrate NNMT inhibitor MS2734 (6) and characterization of this inhibitor in biochemical, biophysical, kinetic, and structural studies. Importantly, we obtained the first crystal structure of human NNMT in complex with a small-molecule inhibitor. The structure of the NNMT-6 complex has unambiguously demonstrated that 6 occupied both substrate and cofactor binding sites. The findings paved the way for developing more potent and selective NNMT inhibitors in the future. [ABSTRACT FROM AUTHOR]

SMYD2 is a lysine methyltransferase that catalyzes the monomethylation of several protein substrates including p53. SMYD2 is overexpressed in a significant percentage of esophageal squamous primary carcinomas, and that overexpression correlates with poor patient survival. However, the mechanism(s) by which SMYD2 promotes oncogenesis is not understood. A small molecule probe for SMYD2 would allow for the pharmacological dissection of this biology. In this report, we disclose LLY-507, a cell-active, potent small molecule inhibitor of SMYD2. LLY-507 is >100-fold selective for SMYD2 over a broad range of methyltransferase and non-methyltransferase targets. A 1.63-Å resolution crystal structure of SMYD2 in complex with LLY-507 shows the inhibitor binding in the substrate peptide binding pocket. LLY-507 is active in cells as measured by reduction of SMYD2-induced monomethylation of p53 Lys370 at submicromolar concentrations. We used LLY-507 to further test other potential roles of SMYD2. Mass spectrometry-based proteomics showed that cellular global histone methylation levels were not significantly affected by SMYD2 inhibition with LLY-507, and subcellular fractionation studies indicate that SMYD2 is primarily cytoplasmic, suggesting that SMYD2 targets a very small subset of histones at specific chromatin loci and/or non-histone substrates. Breast and liver cancers were identified through in silico data mining as tumor types that display amplification and/or overexpression of SMYD2. LLY-507 inhibited the proliferation of several esophageal, liver, and breast cancer cell lines in a dose-dependent manner. These findings suggest that LLY-507 serves as a valuable chemical probe to aid in the dissection of SMYD2 function in cancer and other biological processes. [ABSTRACT FROM AUTHOR]

Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKα (α1 and α2) and ChoKβ isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKα1 and ChoKβ isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKα1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKβ was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKα isoforms (α1 and α2) compared with ChoKβ. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKα2 (equivalent to Leu-419 of ChoKα1), or the corresponding residue Phe-352 of ChoKβ, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation. [ABSTRACT FROM AUTHOR]

Structural genomics and substrate screening provide "chemical fingerprints" and insights into substrate promiscuity for the human family of drug- and hormone-metabolizing cytosolic sulfotransferase enzymes. [ABSTRACT FROM AUTHOR]

Motivation: In the present work we combine computational analysis and experimental data to explore the extent to which binding site similarities between members of the human cytosolic sulfotransferase family correlate with small-molecule binding profiles. Conversely, from a small-molecule point of view, we explore the extent to which structural similarities between small molecules correlate to protein binding profiles.Results: The comparison of binding site structural similarities and small-molecule binding profiles shows that proteins with similar small-molecule binding profiles tend to have a higher degree of binding site similarity but the latter is not sufficient to predict small-molecule binding patterns, highlighting the difficulty of predicting small-molecule binding patterns from sequence or structure. Likewise, from a small-molecule perspective, small molecules with similar protein binding profiles tend to be topologically similar but topological similarity is not sufficient to predict their protein binding patterns. These observations have important consequences for function prediction and drug design.Contact:rafael.najmanovich@ebi.ac.uk [ABSTRACT FROM AUTHOR]

Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl-lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4-dimethylated histone H3 tail as well as for global and gene-specific K4 trimethylation. Here we report the crystal structures of full-length WDR5, WDR5Δ23 and its complexes with unmodified, mono-, di- and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water-mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes. [ABSTRACT FROM AUTHOR]

Although the structures of mammalian cytosolic and mitochondrial ALDH have been determined, several differences, mainly functional, between these two 70% identical isozymes remain unexplained. A major difference is the differential effect of Mg2+ ions that inhibits the cytosolic and activates the mitochondrial isozyme. Here, we have investigated the effect of Mg2+ ions on each individual kinetic step of ALDH1 and ALDH2. The metal ions were found not to affect either acylation or hydride transfer for either isozyme. The lack of a Mg2+ ion effect on hydride transfer was further demonstrated with an E399Q mutant of ALDH1 whose rate-limiting step had been changed from NADH dissociation to hydride transfer. The other steps, however, were affected by Mg2+ ions for both isozymes. The metal ions inhibited NADH dissociation, the rate-limiting step for ALDH1, and enhanced deacylation, the rate- limiting step for ALDH2. Our results indicated that, with both isozymes, Mg2+ ions tightened the binding of NADH, and by binding to the coenzyme, they increased the nucleophilicity of the nucleophile Cys302. The inhibition of ALDH1 and activation of ALDH2 at pH 7.4 are due to their different rate-limiting steps. Mg2+ ions affected similarly the NADH activation of the esterase reaction for both isozymes. In contrast, the metal ions affected only the NAD+ activation of ALDH1. This latter finding and other features described here can be rationalized on the basis of the known three-dimensional structures of the isozymes. [ABSTRACT FROM AUTHOR]

Investigates the interaction between nicotinamide-adenine dinucleotide (NAD) and NAD(P)H dehydrogenases and aldehyde dehydrogenase (ALDH). Features of ALDH; Utilization of Rossmann fold in ALDH-coenzyme interaction; Correlation between the alpha-helix of Rossmann fold and NAD phosphates.

Crystal structures of the L3MBTL1 MBT repeats in complex with histone H4 peptides dimethylated on Lys20 (H4K20me2) show that only the second of the three MBT repeats can bind mono- and dimethylated histone peptides. Its binding pocket has similarities to that of 53BP1 and is able to recognize the degree of histone lysine methylation. An unexpected mode of peptide-mediated dimerization suggests a possible mechanism for chromatin compaction by L3MBTL1. [ABSTRACT FROM AUTHOR]

A correction to the article " L3MBTL1 recognition of mono-and dimethylated histones" that was published in the December 18, 2007 is presented.

A correction to the article "Structural and Chemical Profiling of the Human Cytosolic Sulfotransferases," by Abdellah Allali-Hassani, Wang Pan, Ludmila Dombrovski, Rafi Najmanovich, Wolfram Tempel, Aiping Dong, Peter Loppnau, Fernando Martin, Janet Thornton, Aled Edwards, Alexey Bochkarev, Alexander Plotnikov, Masoud Vedadi and Cheryl Arrowsmith that was published in a previous issue of the journal is presented.

WDR5 (WD40 repeat protein 5) is an essential component of the human trithorax-like family of SET1 [Su(var)3-9 enhancerof- zeste trithorax 1] methyltransferase complexes that carry out trimethylation of histone 3 Lys4 (H3K4me3), play key roles in development and are abnormally expressed in many cancers. In the present study, we showthat the interaction betweenWDR5and peptides from the catalytic domain of MLL (mixed-lineage leukaemia protein) (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis showthat this antagonist binds in the WDR5 peptide-binding pocket with a Kd of 450 nM and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukaemias or other cancers. [ABSTRACT FROM AUTHOR]

Summary: PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1–24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1–24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets. [Copyright &y& Elsevier]

Protein lysine methyltransferase G9a plays key roles in the transcriptional repression of a variety of genes via dimethylation of lysine 9 on histone H3 (H3K9me2) of chromatin as well as dimethylation of nonhistone proteins including tumor suppressor p53. We previously reported the discovery of UNC0321 (3), the most potent G9a inhibitor to date, via structure-based design and structure–activity relationship (SAR) exploration of the quinazoline scaffold represented by BIX01294 (1). Despite its very high in vitro potency, compound 3lacks sufficient cellular potency. The design and synthesis of several generations of new analogues aimed at improving cell membrane permeability while maintaining high in vitro potency resulted in the discovery of a number of novel G9a inhibitors such as UNC0646 (6) and UNC0631 (7) with excellent potency in a variety of cell lines and excellent separation of functional potency versus cell toxicity. The design, synthesis, and cellular SAR of these potent G9a inhibitors are described. [ABSTRACT FROM AUTHOR]

Abstract: Hundreds of genomes have been successfully sequenced to date, and the data are publicly available. At the same time, the advances in large-scale expression and purification of recombinant proteins have paved the way for structural genomics efforts. Frequently, however, little is known about newly expressed proteins calling for large-scale protein characterization to better understand their biochemical roles and to enable structure–function relationship studies. In the Structural Genomics Consortium (SGC), we have established a platform to characterize large numbers of purified proteins. This includes screening for ligands, enzyme assays, peptide arrays and peptide displacement in a 384-well format. In this review, we describe this platform in more detail and report on how our approach significantly increases the success rate for structure determination. Coupled with high-resolution X-ray crystallography and structure-guided methods, this platform can also be used toward the development of chemical probes through screening families of proteins against a variety of chemical series and focused chemical libraries. [Copyright &y& Elsevier]

Background: Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. Methodology/Principal Findings: We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. Conclusions: We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery. Enhanced version: This article can also be viewed as an enhanced version (http://plosone.org/enhanced/pone.0012736) in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. [ABSTRACT FROM AUTHOR]

Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand- and receptor-expressing cells. Consequently, the ephrins and ephrin receptor tyrosine kinases often mediate processes involving cell–cell contact, including cellular adhesion or repulsion, developmental remodeling and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small-cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and is suitable for co-crystallization studies. Complex structures show multiple contacts between kinase and substrates; in particular, two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes. [ABSTRACT FROM AUTHOR]

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 ha HDACs and demonstrate significant differences with previously reported class I and class lib-like HDAC structures. We show that cdHDAC7 has an additional class ha HDAC-speciflc 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 cata- lytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class ha HDACs and facilitate the design of specific modulators. [ABSTRACT FROM AUTHOR]

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins. [ABSTRACT FROM AUTHOR]

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones. [ABSTRACT FROM AUTHOR]

[Display omitted] The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP–TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF , Cyr61 , Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF , Cyr61 , Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells. [ABSTRACT FROM AUTHOR]

Lysine acetyltransferases (KATs) are key mediators of cell signaling. Methods capable of providing new insights into their regulation thus constitute an important goal. Here we report an optimized platform for profiling KAT--ligand interactions in complex proteomes using inhibitor-functionalized capture resins. This approach greatly expands the scope of KATs, KAT complexes, and CoA-dependent enzymes accessible to chemoproteomic methods. This enhanced profiling platform is then applied in the most comprehensive analysis to date of KAT inhibition by the feedback metabolite CoA. Our studies reveal that members of the KAT superfamily possess a spectrum of sensitivity to CoA and highlight NAT 10 as a novel KAT that may be susceptible to metabolic feedback inhibition. This platform provides a powerful tool to define the potency and selectivity of reversible stimuli, such as small molecules and metabolites, that regulate KAT-dependent signaling. [ABSTRACT FROM AUTHOR]

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Kiapp = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7 -- and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, 5-adeno-sylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent WICF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a drug-gable target class. [ABSTRACT FROM AUTHOR]

Epigenetic regulation is involved in numerous physiological and pathogenic processes. Among the key regulators that orchestrate epigenetic signaling are over 50 human protein lysine methyltransferases (PKMTs). Interrogation of the functions of individual PKMTs can be facilitated by target-specific PKMT inhibitors. Given the emerging need for such small molecules, we envisioned an approach to identify target-specific methyltransferase inhibitors by screening privileged small-molecule scaffolds against diverse methyltransferases. In this work, we demonstrated the feasibility of such an approach by identifying the inhibitors of SETD2. N-propyl sinefungin (Pr-SNF) was shown to interact preferentially with SETD2 by matching the distinct transition-state features of SETD2s catalytically active conformer. With Pr- SNF as a structure probe, we further revealed the dual roles of SETD2's post-SET loop in regulating substrate access through a distinct topological reconfiguration. Privileged sinefungin scaffolds are expected to have broad use as structure and chemical probes of methyltransferases. [ABSTRACT FROM AUTHOR]

Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme's hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrate's unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function. [ABSTRACT FROM AUTHOR]

YjeE is an essential ATPase in Escherichia coli whose cellular function remains uncharacterized. Using a genomic library, we have identified rstA as a multicopy suppressor of a conditional yjeE deletion strain. High-copy rstA is the first recorded suppressor for a lesion in yjeE, and this newly charted genetic interaction has the potential to be informative about the function, with further study of the interacting partners. [ABSTRACT FROM AUTHOR]

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation. [ABSTRACT FROM AUTHOR]

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation. [ABSTRACT FROM AUTHOR]

Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16INK4A, when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression. [ABSTRACT FROM AUTHOR]

Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways. [ABSTRACT FROM AUTHOR]

SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. [ABSTRACT FROM AUTHOR]

Background: The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats. Methodology/Principal Findings: We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 Å resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a ''cavity insertion recognition mode'' to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1]. Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation. Conclusions: The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage. Enhanced version: This article can also be viewed as an enhanced version (http://plosone.org/enhanced/pone.0007274/) in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. [ABSTRACT FROM AUTHOR]

Summary: Ephrin receptors (Eph) affect cell shape and movement, unlike other receptor tyrosine kinases that directly affect proliferative pathways. The kinase domain of EphA3 is activated by ephrin binding and receptor oligomerization. This activation is associated with two tyrosines in the juxtamembrane region; these tyrosines are sites of autophosphorylation and interact with the active site of the kinase to modulate activity. This allosteric event has important implications both in terms of understanding signal transduction pathways mediated by Eph kinases as well as discovering specific therapeutic ligands for receptor kinases. In order to provide further details of the molecular mechanism through which the unphosphorylated juxtamemebrane region blocks catalysis, we studied wild-type and site-specific mutants in detail. High-resolution structures of multiple states of EphA3 kinase with and without the juxtamembrane segment allowed us to map the coupled pathway of residues that connect the juxtamembrane segment, the activation loop, and the catalytic residues of the kinase domain. This highly conserved set of residues likely delineates a molecular recognition pathway for most of the Eph RTKs, helping to characterize the dynamic nature of these physiologically important enzymes. [Copyright &y& Elsevier]

Summary: Sirtuins are NAD+-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD+-dependent deacetylase activity with an IC50 value of 22 μM. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD+, the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors. [Copyright &y& Elsevier]

To gain a better insight into the alterations of brain function after chronic ethanol, we measured the release of various neurotransmitters from nerve terminals of cortex and hippocampus isolated from rats chronically fed with ethanol. The K[SUP+]-evoked release of [SUP3]H]acetylcholine (ACh), [ [SUP3]H]dopamine (DA), [SUP3]H]glutamate (Glu) and [SUP3]H]noradrenaline (NA) was determined in superfused synaptosomes of brain cortex and hippocampus from rats exposed to the Lieber-DeCarli alcohol liquid diet for 5 weeks. In cortical synaptosomes, chronic ethanol administration did not affect the release of ACh and of DA, while significantly decreasing the release of Glu and NA. The endogenous levels of NA, DA and their metabolites were unchanged. In hippocampal synaptosomes the only effect of chronic alcohol was an increased release of Glu. It can be concluded that at presynaptic level chronic ethanol alters brain neurotransmitter systems selectively. Glutamatergic and noradrenergic nerve terminals from cortex are more vulnerable than those from hippocampus. [ABSTRACT FROM AUTHOR]

Human stomach mucosa contains a characteristic alcohol dehydrogenase (ADH) enzyme, σσADH. Its cDNA has been cloned from a human stomach library and sequenced. The deduced amino acid sequence shows 59-70% identities with the other human ADH classes, demonstrating that the stomach enzyme represents a distinct structure, constituting class IV, coded by a separate gene. ADH7. The amino acid identity with the rat stomach class IV ADH is 88%, which is intermediate between constant and variable dehydrogenases. This value reflects higher conservation than for the classical liver enzymes of class I, compatible with a separate functional significance of the class IV enzyme. Its enzymic features can be correlated with its structural characteristics. The residues lining the substrate-binding cleft are bulky and hydmphobic, similar to those of the class I enzyme: this explains the similar specificity of both classes, compatible with the origin of class IV from class I. Position 47 has Arg, in contrast to Gly in the rat class IV enzyme, bm this Arg is still associated with an extremely high activity (kcat = 1510 min-1) and weak coenzyme binding (KiaNAD+ = 1.6 mM). Thus, the strong interaction with coenzyme imposed by Arg47 in class I is probably compensated for in crass IV by changes that may negatively affect coenzyme binding: Glu230, His271, Ash260, Asn261, Asn363. The still higher activity and weaker coenzyme binding of rat class IV (kcat = 2600 min-1, KiaNAD = 4 mM) can be correlated to the exchanges to Gly47, Gln230 and Tyr363. An important change at position 294, with Val in human and Ala in rat class IV, is probably responsible for the dramatic difference in Km values for ethanol between human (37 mM) and rat (2.4 M) class IV enzymes. [ABSTRACT FROM AUTHOR]

A correction to the article "A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells," by Masoud Vedadi and colleagues that was published online in the July 10, 2011 issue is presented.