The structure of Staphylococcus aureus epidermolytic toxin A, an atypic serine protease, at 1.7 Å resolution

AbstractBackground: Staphylococcal epidermolytic toxins A and B (ETA and ETB) are responsible for the staphylococcal scalded skin syndrome of newborn and young infants; this condition can appear just a few hours after birth. These toxins cause the disorganization and disruption of the region between the stratum spinosum and the stratum granulosum —  two of the three cellular layers constituting the epidermis. The physiological substrate of ETA is not known and, consequently, its mode of action in vivo remains an unanswered question. Determination of the structure of ETA and its comparison with other serine proteases may reveal insights into ETA's catalytic mechanism.Results: The crystal structure of staphylococcal ETA has been determined by multiple isomorphous replacement and refined at 1.7 Å resolution with a crystallographic R factor of 0.184. The structure of ETA reveals it to be a new and unique member of the trypsin-like serine protease family. In contrast to other serine protease folds, ETA can be characterized by ETA-specific surface loops, a lack of cysteine bridges, an oxyanion hole which is not preformed, an S1 specific pocket designed for a negatively charged amino acid and an ETA-specific N-terminal helix which is shown to be crucial for substrate hydrolysis.Conclusions: Despite very low sequence homology between ETA and other trypsin-like serine proteases, the ETA crystal structure, together with biochemical data and site-directed mutagenesis studies, strongly confirms the classification of ETA in the Glu-endopeptidase family. Direct links can be made between the protease architecture of ETA and its biological activity

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate

PLoS Pathog

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical alpha/beta HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens

Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins

E6 viral oncoproteins are key players in epithelial tumors induced by papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. We solved the crystal structures of bovine (BPV1) and human (HPV16) papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively. In both E6 proteins, two zinc domains and a linker helix form a basic-hydrophobic pocket, which captures helical LxxLL motifs in a way compatible with other interaction modes. Mutational inactivation of the LxxLL binding pocket disrupts the oncogenic activities of both E6 proteins. This work reveals the structural basis of both the multifunctionality and the oncogenicity of E6 proteins

Etude cristallographique d'un complexe nucleoproteique forme entre l'aspartyl-tRNA synthetase de levure et l'acide ribonucleique de transfert specifique de l'acide aspartique

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Étude structurale de l'histoneméthyltransférase CARM1 et de ses complexes biologiquement significatifs (des structures 3D vers la conception rationnelle de composés à action pharmacologique)

Les "protéine arginine méthyltransférases" (PRMT) sont impliquées dans de nombreux processus cellulaires : transcription, maturation et transport des ARN, traduction, transduction du signal, réplication et réparation de l'ADN, et apoptose. Différents travaux ont montré que des dérégulations de ces mécanismes impliquant les PRMT peuvent induire certains cancers, faisant de ces enzymes de nouvelles cibles potentielles en chimiothérapie. Il s avère donc crucial de comprendre le mode d action des PRMT à l échelle atomique, à la fois au niveau fondamental et pour le développement de nouveaux médicaments. Les travaux décrits ici s intéressent à la protéine PRMT4/CARM1 et s appuient sur des études structurales par bio-cristallographie, pour comprendre les mécanismes de la réaction de méthylation catalysée par CARM1 et découvrir des inhibiteurs spécifiques, mais aussi sur des études en solution, pour caractériser l interaction entre CARM1 et ses substrats.Protein arginine methyltransferases (PRMTs) are involved in several cellular mechanisms: transcription, RNA maturation and transport, translation, signal transduction, DNA replication and repair, and apoptosis. Different studies showed that deregulation of those mechanisms involving PRMTs can induce some cancers, making these enzymes new potential targets for chemotherapy. It is therefore crucial to understand the mode of action of PRMTs at the atomic scale, both at the fundamental level and for the development of new drugs. The studies described here focus on PRMT4/CARM1 and rely on structural studies by bio-crystallography, in order to understand the methylation mechanisms catalyzed by CARM1 and to discover specific inhibitors, but also on in vitro studies, to characterize the interaction between CARM1 and its substrates.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF

ETUDE STRUCTURALE DU SYSTEME ARGINYL-ARNT SYNTHETASE CHEZ LA LEVURE SACCHAROMYCES CEREVISIAE

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Etude structurale de la Leucyl-ARNt synthétase d'Aquifex aeolicus

STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Etude structurale et fonctionnelle de la protéine arginine méthyltransférase CARM1

La protéine CARM1 "Coactivator-Associated aRginine Methyltransferase 1" a été initialement identifiée pour sa fonction coactivatrice de la transcription impliquant différents récepteurs nucléaires des hormones. Cette voie d activation de la transcription est un processus régulé par plusieurs étapes et différentes cascades de signalisation impliquant de nombreux corégulateurs. Dans ce contexte, deux cibles privilégiées ont été identifiées pour l'activité de méthylation de CARM1: l'histone H3 et la protéine CBP. CARM1 est également impliquée dans d autres processus, comme la maturation des ARN et la transmission du signal cellulaire. Une dérégulation de ces différents processus pouvant induire certains cancers, CARM1 constitue une des nouvelles cibles potentielles en chimiothérapie. Les résultats obtenus durant cette thèse sont répartis en quatre chapitres. Le chapitre 3 présente les structures cristallographiques de domaines isolés de CARM1 résolues seules ou en complexe avec différents cofacteurs. Le chapitre 4 porte sur les études fonctionnelles en solution par la comparaison de différentes constructions et de mutants ponctuels. Ces études mettent en évidences des zones et des résidus cruciaux à l activité de CARM1. Le chapitre 5 détaille l étude des interactions entre CARM1 et différents substrats. Ceci a été fait par différentes approches : l étude de protéines complètes (histone H3 et CBP), de fragments de ces protéines et une approche qui consiste à fixer de façon covalente des peptides de l histone H3 à un complexe binaire CARM1-cofacteur. Le chapitre 6 porte sur la recherche d inhibiteurs spécifiques de CARM1, travaux effectués dans le cadre de collaborations.The protein CARM1 "Coactivator-Associated aRginine Methyltransferase 1" was initially identified for its activity to coactivate transcription under the regulation of some nuclear receptors. This kind of activation process is regulated by a large number of coactivators involved at different stages. In this context, CARM1 was identified to methylates two major targets: histone H3 and CBP. CARM1 is also involved in other biological events like RNA processing and signal transmission. Disturbances of these different processes can induce cancers and CARM1 is by the way a new potential pharmacological target in chemotherapy. The results obtained during this thesis work are divided in four chapters. Chapter 3 presents the crystallographic structures of isolated domains of CARM1, alone or in complex with different cofactors. Chapter 4 describes the functional studies in solution by comparisons of different constructions and mutant forms. These studies highlight the crucial role of different areas and residues for the CARM1 activity. Chapter 5 details studies of interactions between CARM1 and selected target substrates (histone H3 and CBP). It has been done by three different approaches: interactions with full length proteins, fragments of these proteins and an approach consisting of covalently bind H3 peptides to a binary complex CARM1-cofactor. Chapter 6 presents a collaborative work done to discover CARM1 specific inhibitors.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF