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This protocol describes the proteomics technique called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis or SIESTA 1,2. SIESTA can be used for universal discovery of enzyme substrates that shift in thermal stability or solubility upon post-translational modification (PTM). Experimental design, proteomics sample preparation and data analysis are the key stages of this protocol. Data analysis can be performed using our SIESTA package hosted on GitHub 3. When performed with classical thermal proteome profiling (TPP), the protocol will take 5 days for sample preparation and 14 days of sample analysis by mass spectrometry (the current protocol). If our high-throughput version of TPP called Proteome Integral Solubility Alteration assay (PISA) 4 is used instead, the sample analysis time by mass spectrometry is reduced to 1-2 days for the same number of conditions.

Despite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery., The global identification of enzyme substrates is still challenging. Here, the authors develop a method based on proteome-wide thermal shift assays to discover enzyme substrates directly from cell lysates, identifying known and novel oxidoreductase, kinase and poly-(ADP-ribose) polymerase substrates.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions., ADP-ribose binding macro domains facilitate the enrichment and detection of cellular ADP-ribosylation. Here, the authors generate an engineered macro domain with increased ADP-ribose affinity, improving the identification of ADP-ribosylated proteins by proteomics, western blot and immunofluorescence.

Pseudomonasare a common cause of hospital acquired infections that may be lethal. ADP-ribosyltransferase activities ofPseudomonasexotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, a hydrophobic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive novel binding interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the hydrophobic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the hydrophobic C-terminus with a fragment fromVibrioVis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the hydrophobic C-terminal segment.Short summaryCrystal structures of Pseudomonas exotoxins-S and –T identify a novel hydrophobic interface with 14-3-3 proteins, and we show that 14-3-3 activates these toxins independent of their phosphopeptide groove binding C-termini, by preventing their aggregation.

A series of (Z)-4-(3-carbamoylphenylamino)-4-oxobut-2-enyl amides were synthesized and tested for their ability to inhibit the mono-(ADP-ribosyl)transferase, PARP14 (a.k.a. BAL-2; ARTD-8). Two synthetic routes were established for this series and several compounds were identified as sub-micromolar inhibitors of PARP14, the most potent of which was compound 4t, IC50=160nM. Furthermore, profiling other members of this series identified compounds with >20-fold selectivity over PARP5a/TNKS1, and modest selectivity over PARP10, a closely related mono-(ADP-ribosyl)transferase.

Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins might change their thermal stability. SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.

A series of diaryl ethers were designed and synthesized to discern the structure activity relationships against the two closely related mono-(ADP-ribosyl)transferases PARP10 and PARP14. Structure activity studies identified 8b as a sub-micromolar inhibitor of PARP10 with ∼15-fold selectivity over PARP14. In addition, 8k and 8m were discovered to have sub-micromolar potency against PARP14 and demonstrated moderate selectivity over PARP10. A crystal structure of the complex of PARP14 and 8b shows binding of the compound in a novel hydrophobic pocket and explains both potency and selectivity over other PARP family members. In addition, 8b, 8k and 8m also demonstrate selectivity over PARP1. Together, this study identified novel, potent and metabolically stable derivatives to use as chemical probes for these biologically interesting therapeutic targets.

The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD(+) binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity.

Summary Poly-ADP-ribose polymerases (PARPs1-16) play pivotal roles in diverse cellular processes. PARPs that catalyze poly-ADP-ribosylation (PARylation) are the best characterized PARP family members because of the availability of potent and selective inhibitors for these PARPs. There has been comparatively little success in developing selective small-molecule inhibitors of PARPs that catalyze mono-ADP-ribosylation (MARylation), limiting our understanding of the cellular role of MARylation. Here we describe the structure-guided design of inhibitors of PARPs that catalyze MARylation. The most selective analog, ITK7, potently inhibits the MARylation activity of PARP11, a nuclear envelope-localized PARP. ITK7 is greater than 200-fold selective over other PARP family members. Using live-cell imaging, we show that ITK7 causes PARP11 to dissociate from the nuclear envelope. These results suggest that the cellular localization of PARP11 is regulated by its catalytic activity.

Searching for selective tankyrases (TNKSs) inhibitors, a new small series of 6,8-disubstituted triazolo[4,3-b]piridazines has been synthesized and characterized biologically. Structure-based optimization of the starting hit compound NNL (3) prompted us to the discovery of 4-(2-(6-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-ylamino)ethyl)-phenol (12), a low nanomolar selective TNKSs inhibitor working as NAD isostere as ascertained by crystallographic analysis. Preliminary biological data candidate this new class of derivatives as a powerful pharmacological tools in the unraveling of TNKS implications in physiopathological conditions.

Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

ADP-ribosylation of proteins regulates protein activities in various processes including transcription control, chromatin organization, organelle assembly, protein degradation, and DNA repair. Modulating the proteins involved in the metabolism of ADP-ribosylation can have therapeutic benefits in various disease states. Protein crystal structures can help understand the biological functions, facilitate detailed analysis of single residues, as well as provide a basis for development of small molecule effectors. Here we review recent advances in our understanding of the structural biology of the writers, readers, and erasers of ADP-ribosylation.

Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

Poly(ADP-ribose) polymerases (PARPs) are key enzymes in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, and suffer from poor selectivity. PARP14 has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Herein, we describe a small molecule microarray-based strategy for high-throughput synthesis, screening of >1000 potential bidentate inhibitors of PARPs, and the successful discovery of a potent PARP14 inhibitor H10 with >20-fold selectivity over PARP1. Co-crystallization of the PARP14/H10 complex indicated H10 bound to both the nicotinamide and the adenine subsites. Further structure-activity relationship studies identified important binding elements in the adenine subsite. In tumor cells, H10 was able to chemically knockdown endogenous PARP14 activities.

The diphtheria toxin-like ADP-ribosyltransferases (ARTDs) are an enzyme family that catalyzes the transfer of ADP-ribose units onto substrate proteins by using nicotinamide adenine dinucleotide (NAD(+)) as a cosubstrate. They have a documented role in chromatin remodelling and DNA repair, and inhibitors of ARTD1 and 2 (PARP1 and 2) are currently in clinical trials for the treatment of cancer. The detailed function of most other ARTDs is still unknown. By using virtual screening, we identified small ligands of ARTD7 (PARP15/BAL3) and ARTD8 (PARP14/BAL2). Thermal-shift assays confirmed that 16 compounds, belonging to eight structural classes, bound to ARTD7/ARTD8. Affinity measurements with isothermal titration calorimetry for two isomers of the most promising hit compound confirmed binding in the low micromolar range to ARTD8. Crystal structures showed anchoring of the hits in the nicotinamide pocket. These results form a starting point in the development of chemical tools for the study of the role and function of ARTD7 and ARTD8.

ADP-ribosylation is involved in the regulation of DNA repair, transcription, and other processes. The 18 human ADP-ribose transferases with diphtheria toxin homology include ARTD1/PARP1, a cancer drug target. Knowledge of other family members may guide therapeutics development and help evaluate potential drug side effects. Here, we present the crystal structure of human ARTD15/PARP16, a previously uncharacterized enzyme. ARTD15 features an α-helical domain that packs against its transferase domain without making direct contact with the NAD+-binding crevice or the donor loop. Thus, this novel domain does not resemble the regulatory domain of ARTD1. ARTD15 displays auto-mono(ADP-ribosylation) activity and is affected by canonical poly(ADP-ribose) polymerase inhibitors. These results add to a framework that will facilitate research on a medically important family of enzymes. Background: ADP-ribose transferases ARTD1–3/PARP1–3 have an α-helical domain that closes over the NAD+-binding site. Results: Human ARTD15/PARP16 is a mono(ADP-ribose) transferase with a novel α-helical domain that interacts with a catalytic domain loop. Conclusion: The ARTD15 transferase domain is likely regulated by effector binding to the adjacent helical domain. Significance: This provides a basis for understanding the enzymatic mechanism of this previously uncharacterized enzyme.

Inhibitors of poly-ADP-ribose polymerase (PARP) family proteins are currently in clinical trials as cancer therapeutics, yet the specificity of many of these compounds is unknown. Here we evaluated a series of 185 small-molecule inhibitors, including research reagents and compounds being tested clinically, for the ability to bind to the catalytic domains of 13 of the 17 human PARP family members including the tankyrases, TNKS1 and TNKS2. Many of the best-known inhibitors, including TIQ-A, 6(5H)-phenanthridinone, olaparib, ABT-888 and rucaparib, bound to several PARP family members, suggesting that these molecules lack specificity and have promiscuous inhibitory activity. We also determined X-ray crystal structures for five TNKS2 ligand complexes and four PARP14 ligand complexes. In addition to showing that the majority of PARP inhibitors bind multiple targets, these results provide insight into the design of new inhibitors.

RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long alpha-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5'-triphosphate, cytidine 5'-triphosphate, and uridine 5'-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.

We report two crystal structures of the PARP domain of human tankyrase-2 (TNKS2). Tankyrases are involved in fundamental cellular processes such as telomere homeostasis and Wnt signaling. The complex of TNKS2 with the potent inhibitor XAV939 provides insights into the molecular basis of the strong interaction and suggests routes for further development of tankyrase inhibitors.

Poly(ADP-ribose) polymerases (PARPs) activate DNA repair mechanisms upon stress- and cytotoxin-induced DNA damage, and inhibition of PARP activity is a lead in cancer drug therapy. We present a structural and functional analysis of the PARP domain of human PARP-3 in complex with several inhibitors. Of these, KU0058948 is the strongest inhibitor of PARP-3 activity. The presented crystal structures highlight key features for potent inhibitor binding and suggest routes for creating isoenzyme-specific PARP inhibitors.

DEXD/H-box RNA helicases couple ATP hydrolysis to RNA remodeling by an unknown mechanism. We used x-ray crystallography and biochemical analysis of the human DEXD/H-box protein DDX19 to investigate its regulatory mechanism. The crystal structures of DDX19, in its RNA-bound prehydrolysis and free posthydrolysis state, reveal an alpha-helix that inserts between the conserved domains of the free protein to negatively regulate ATPase activity. This finding was corroborated by biochemical data that confirm an autoregulatory function of the N-terminal region of the protein. This is the first study describing crystal structures of a DEXD/H-box protein in its open and closed cleft conformations.

Impairment of the formation or action of hydrogen sulfide (H(2)S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine beta-synthase and cystathionine gamma-lyase (CSE) are two pyridoxal-5'-phosphate (PLP)-dependent enzymes largely responsible for the production of H(2)S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP.PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H(2)S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE.PLP.PAG complex highlights the particular importance of Tyr(114) in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of ferrochelatase and a catalytic antibody with ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of ferrochelatase in controlling stereospecificity, distortion and metallation.

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.

Argininosuccinate synthetase catalyzes the transformation of citrulline and aspartate into argininosuccinate and pyrophos­phate using the hydrolysis of ATP to AMP and pyrophos­phate. This enzymatic process constitutes the rate-limiting step in both the urea and arginine–citrulline cycles. Previous studies have investigated the crystal structures of argininosuccinate synthetase from bacterial species. In this work, the first crystal structure of human argininosuccinate synthetase in complex with the substrates citrulline and aspartate is presented. The human enzyme is compared with structures of argininosuccinate synthetase from bacteria. In addition, the structure also provides new insights into the function of the numerous clinical mutations identified in patients with type I citrullinaemia (also known as classic citrullinaemia).

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.

Sister chromatid cohesion, formed by the cohesin protein complex, is essential for chromosome segregation. In order for cohesion to be established, the cohesin subunit SMC3 needs to be acetylated by a homolog of the ESCO1/Eco1 acetyltransferases, the enzymatic mechanism of which has remained unknown. Here we report the crystal structure of the ESCO1 acetyltransferase domain in complex with acetyl-coenzyme A, and show by SAXS that ESCO1 is a dimer in solution. The structure reveals an active site that lacks a potential catalytic base side chain. However, mutation of glutamate 789, a surface residue that is close to the automodification target lysine 803, strongly reduces autoacetylation of ESCO1. Moreover, budding yeast Smc3 mutated at the conserved residue D114, adjacent to the cohesion-activating acetylation site K112,K113, cannot be acetylated in vivo. This indicates that ESCO1 controls cohesion through substrate-assisted catalysis. Thus, this study discloses a key mechanism for cohesion establishment.

The polyamine synthesis enzyme spermidine synthase (SPDS) has been cloned from the model nematode Caenorhabditis elegans. Biochemical characterisation of the recombinantly expressed protein revealed a high degree of similarity to other eukaryotic SPDS with the exception of a low affinity towards the substrate decarboxylated S-adenosylmethionine (Km = 110 microM) and a less pronounced feedback inhibition by the second reaction product 5'-methylthioadenosine (IC50 = 430 microM). The C. elegans protein that carries a nematode-specific insertion of 27 amino acids close to its N-terminus was crystallized, leading to the first X-ray structure of a dimeric eukaryotic SPDS.

Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.

Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway. It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX. The crystal structures of ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work. The enzyme is a homodimer, with clear asymmetry between the monomers with regard to the porphyrin binding cleft and the mode of metal binding. The Co(II) and Cd(II) complexes reveal the metal binding site which consists of the invariant amino acids H235, E314, and S275 and solvent molecules. The shortest distance to the metal reveals that amino acid H235 is the primary metal binding residue. A second site with bound Cd(II) was found close to the surface of the molecule, approximately 14 A from H235, with E97, H317, and E326 participating in metal coordination. It is suggested that this site corresponds to the magnesium binding site in Bacillus subtilis ferrochelatase. The latter site is also located at the surface of the molecule and thought to be involved in initial metal binding and regulation.

The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-N-[1-(pyridin-2-yl)ethyl]propanamide, 1, has previously been identified as a potent but unselective inhibitor of diphtheria toxin-like ADP-ribosyltransferase 3 (ARTD3). Herein we describe synthesis and evaluation of 55 compounds in this class. It was found that the stereochemistry is of great importance for both selectivity and potency and that substituents on the phenyl ring resulted in poor solubility. Certain variations at the meso position were tolerated and caused a large shift in the binding pose. Changes to the ethylene linker that connects the quinazolinone to the amide were also investigated but proved detrimental to binding. By combination of synthetic organic chemistry and structure-based design, two selective inhibitors of ARTD3 were discovered.

Summary ADP-ribosyltransferases (ARTs) catalyze the transfer of ADP-ribose from NAD + onto substrates. Some ARTs generate in an iterative process ADP-ribose polymers that serve as adaptors for distinct protein domains. Other ARTs, exemplified by ARTD10, function as mono-ADP-ribosyltransferases, but it has been unclear whether this modification occurs in cells and how it is read. We observed that ARTD10 colocalized with ARTD8 and defined its macrodomains 2 and 3 as readers of mono-ADP-ribosylation both in vitro and in cells. The crystal structures of these two ARTD8 macrodomains and isothermal titration calorimetry confirmed their interaction with ADP-ribose. These macrodomains recognized mono-ADP-ribosylated ARTD10, but not poly-ADP-ribosylated ARTD1. This distinguished them from the macrodomain of macroH2A1.1, which interacted with poly- but not mono-ADP-ribosylated substrates. Moreover, Ran, an ARTD10 substrate, was also read by ARTD8 macrodomains. This identifies readers of mono-ADP-ribosylated proteins, defines their structures, and demonstrates the presence of this modification in cells.

Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into ( i ) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA's active and inhibitory sites, and ( ii ) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

Ferrochelatase catalyzes the insertion of Fe(2+) into protoporphyrin IX. The enzymatic product heme (protoheme IX) is a well-known cofactor in a wide range of proteins. The insertion of metal ions other than Fe(2+) occurs rarely in vivo, but all ferrochelatases that have been studied can insert Zn(2+) at a good rate in vitro. Co(2+), but not Cu(2+), is known to be a good substrate of the mammalian and Saccharomyces cerevisiae ferrochelatases. In contrast, Cu(2+), but not Co(2+), has been found to be a good substrate of bacterial Bacillus subtilis ferrochelatase. It is not known how ferrochelatase discriminates between different metal ion substrates. Structural analysis of B. subtilis ferrochelatase has shown that Tyr13 is an indirect ligand of Fe(2+) and a direct ligand of a copper mesoporphyrin product. A structure-based comparison revealed that Tyr13 aligns with a Met residue in the S. cerevisiae and human ferrochelatases. Tyr13 was changed to Met in the B. subtilis enzyme by site-directed mutagenesis. Enzymatic measurements showed that the modified enzyme inserted Co(2+) at a higher rate than the wild-type B. subtilis ferrochelatase, but it had lost the ability to use Cu(2+) as a substrate. Thus, the B. subtilis Tyr13Met ferrochelatase showed the same metal specificity as that of the ferrochelatases from S. cerevisiae and human.

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

Poly-ADP-ribose polymerases (PARPs) catalyze transfer of ADP-ribose from NAD(+) to specific residues in their substrate proteins or to growing ADP-ribose chains. PARP activity is involved in processes such as chromatin remodeling, transcription control, and DNA repair. Inhibitors of PARP activity may be useful in cancer therapy. PARP2 is the family member that is most similar to PARP1, and the two can act together as heterodimers. We used X-ray crystallography to determine two structures of the catalytic domain of human PARP2: the complexes with PARP inhibitors 3-aminobenzamide and ABT-888. These results contribute to our understanding of structural features and compound properties that can be employed to develop selective inhibitors of human ADP-ribosyltransferases.

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins. Enhanced version This article can also be viewed as an enhanced version 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.

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 Å. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations. Enhanced version This article can also be viewed as an enhanced version 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.

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

Ferrochelatase catalyzes the terminal step in the heme biosynthetic pathway, i.e., the incorporation of Fe(II) into protoporphyrin IX. Various biochemical and biophysical methods have been used to probe the enzyme for metal binding residues and the location of the active site. However, the location of the metal binding site and the path of the metal into the porphyrin are still disputed. Using site-directed mutagenesis on Bacillus subtilis ferrochelatase we demonstrate that exchange of the conserved residues His183 and Glu264 affects the metal affinity of the enzyme. We also present the first X-ray crystal structure of ferrochelatase with iron. Only a single iron was found in the active site, coordinated in a square pyramidal fashion by two amino acid residues, His183 and Glu264, and three water molecules. This iron was not present in the structure of a His183Ala modified ferrochelatase. The results strongly suggest that the insertion of a metal ion into protoporphyrin IX by ferrochelatase occurs from a metal binding site represented by His183 and Glu264.