Insights into Chi recognition from the structure of an AddAB-type helicase–nuclease complex

Homologous recombination DNA repair requires double-strand break resection by helicase–nuclease enzymes. The crystal structure of bacterial AddAB in complex with DNA substrates shows that it employs an inactive helicase site to recognize ‘Chi' recombination hotspot sequences that regulate resection

The Measurement of Territorial Differences in the Information Society

Glutamine synthetase (GS, EC 6.3.1.2; also known as γ-glutamyl:ammonia ligase) catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine. The enzyme has essential roles in different tissues and species, which have led to its consideration as a drug or an herbicide target. In this article, we describe studies aimed at the discovery of new antimicrobial agents targeting Mycobacterium tuberculosis, the causative pathogen of tuberculosis. A number of distinct classes of GS inhibitors with an IC50 of micromolar value or better were identified via high-throughput screening. A commercially available purine analogue similar to one of the clusters identified (the diketopurines), 1-[(3,4-dichlorophenyl)methyl]-3,7-dimethyl-8-morpholin-4-yl-purine-2,6-dione, was also shown to inhibit the enzyme, with a measured IC50 of 2.5 ± 0.4 μM. Two X-ray structures are presented: one is a complex of the enzyme with the purine analogue alone (2.55-Å resolution), and the other includes the compound together with methionine sulfoximine phosphate, magnesium and phosphate (2.2-Å resolution). The former represents a relaxed, inactive conformation of the enzyme, while the latter is a taut, active one. These structures show that the compound binds at the same position in the nucleotide site, regardless of the conformational state. The ATP-binding site of the human enzyme differs substantially, explaining why it has an ∼ 60-fold lower affinity for this compound than the bacterial GS. As part of this work, we devised a new synthetic procedure for generating l-(SR)-methionine sulfoximine phosphate from l-(SR)-methionine sulfoximine, which will facilitate future investigations of novel GS inhibitors

Operator Spin Foam Models

The goal of this paper is to introduce a systematic approach to spin foams. We define operator spin foams, that is foams labelled by group representations and operators, as the main tool. An equivalence relation we impose in the set of the operator spin foams allows to split the faces and the edges of the foams. The consistency with that relation requires introduction of the (familiar for the BF theory) face amplitude. The operator spin foam models are defined quite generally. Imposing a maximal symmetry leads to a family we call natural operator spin foam models. This symmetry, combined with demanding consistency with splitting the edges, determines a complete characterization of a general natural model. It can be obtained by applying arbitrary (quantum) constraints on an arbitrary BF spin foam model. In particular, imposing suitable constraints on Spin(4) BF spin foam model is exactly the way we tend to view 4d quantum gravity, starting with the BC model and continuing with the EPRL or FK models. That makes our framework directly applicable to those models. Specifically, our operator spin foam framework can be translated into the language of spin foams and partition functions. We discuss the examples: BF spin foam model, the BC model, and the model obtained by application of our framework to the EPRL intertwiners.Comment: 19 pages, 11 figures, RevTex4.

Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice

Structural Studies of Glutamine Synthetases – Towards the Development of Novel Antitubercular Agents

Glutamine synthetase (GS) plays an important role in nitrogen metabolism, where it catalyzes the ATP-dependent condensation of glutamate and ammonia to yield glutamine. Recent studies showed the importance of M. tuberculosis GS (MtGS) for growth and survival of the bacterium, and demonstrated its potential as a drug target. This thesis presents structural studies of MtGS and mammalian GSs, which are aimed at identifying and developing novel inhibitors against the mycobacterial target. The structure of MtGS was solved in complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium and ADP. The complex structure provides a detailed picture of the active site, offering several insights into catalysis and inhibition, as well as forming a solid basis for structure-based drug design. The apo canine GS and liganded human GS structures described in this thesis represent the first structures of the mammalian enzymes. Comparison of the structures revealed substrate-induced conformational changes. Inspection of the nucleotide-binding site showed that it differs from that of MtGS, thus offering good opportunities to design specific and selective inhibitors of the mycobacterial enzyme. The amino acid-binding site of MtGS was evaluated as a target for inhibition, using a combination of a literature survey, structure-based virtual screening and the synthesis of a small library of compounds. As a result, several new inhibitors of MtGS could be identified. Finally, the structural basis for inhibition of MtGS by a purine analogue (PA) is provided. PA, an analogue of a class of compounds found to inhibit MtGS in a high-throughput screening assay, targets the nucleotide-binding site. The architecture of the HsGS nucleotide-binding site indicates that PA would not be able to bind to the human enzyme, offering good prospects for selective inhibition of MtGS

Crystal Structures of Mammalian Glutamine Synthetases Illustrate Substrate-Induced Conformational Changes and Provide Opportunities for Drug and Herbicide Design

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 Å. 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 Å and 2.6 Å, 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

Structural basis for the inhibition of Mycobacterium tuberculosis glutamine synthetase by novel ATP-competitive inhibitors

Glutamine synthetase (GS, EC 6.3.1.2; also known as γ-glutamyl:ammonia ligase) catalyzes the ATP-dependent condensation of glutamate and ammonia to form glutamine. The enzyme has essential roles in different tissues and species, which have led to its consideration as a drug or an herbicide target. In this article, we describe studies aimed at the discovery of new antimicrobial agents targeting Mycobacterium tuberculosis, the causative pathogen of tuberculosis. A number of distinct classes of GS inhibitors with an IC50 of micromolar value or better were identified via high-throughput screening. A commercially available purine analogue similar to one of the clusters identified (the diketopurines), 1-[(3,4-dichlorophenyl)methyl]-3,7-dimethyl-8-morpholin-4-yl-purine-2,6-dione, was also shown to inhibit the enzyme, with a measured IC50 of 2.5 ± 0.4 μM. Two X-ray structures are presented: one is a complex of the enzyme with the purine analogue alone (2.55-Å resolution), and the other includes the compound together with methionine sulfoximine phosphate, magnesium and phosphate (2.2-Å resolution). The former represents a relaxed, inactive conformation of the enzyme, while the latter is a taut, active one. These structures show that the compound binds at the same position in the nucleotide site, regardless of the conformational state. The ATP-binding site of the human enzyme differs substantially, explaining why it has an ∼ 60-fold lower affinity for this compound than the bacterial GS. As part of this work, we devised a new synthetic procedure for generating l-(SR)-methionine sulfoximine phosphate from l-(SR)-methionine sulfoximine, which will facilitate future investigations of novel GS inhibitors