Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase

Compounds binding to the bacterial beta ring

The present invention relates to compounds which bind to the hydrophobic pocket of the B clamp, i.e., to the surface of the Bring with which said protein interacts with other proteins of the bacterial replication complex during DNA replication. These compounds are derived from the acetylated peptide AcOLDLF (P6) to improve their affinity to their target

Engineering Dion-Jacobson Perovskites in Polariton Waveguides

Hybrid two-dimensional perovskites hold considerable promise as semiconductors for a wide range of optoelectronic applications. Many efforts are addressed to exploit the potential of these materials by tailoring their characteristics. In this work, the optical properties and electronic band structure in three new Dion-Jacobson (DJ) perovskites (PVKs) are engineered by modulating their structural distortion. Two different interlayer cations: 1-6, Hexamethylendiammonium, HE, and 3-(Dimethylamino)-1-propylammonium, DMPA, have been selected to investigate the role of the cation length and the ammonium binding group on the crystalline structure. This study provides new insights into the understanding of the structure-property relationship in DJ perovskites and demonstrates that exciton characteristics can be easily modulated with the judicious design of the organic cations. DJ PVKs developed in this work were also grown as size-controlled single crystal microwires through a microfluidic-assisted synthesis technique and integrated in a nanophotonic device. The DJ PVK microwire acts as a waveguide exhibiting strong light-matter coupling between the crystal optical modes and DJ PVK exciton. Through the investigation of these polariton waveguides, the nature of the double peak emission, which is often observed in these materials and whose nature is largely debated in the literature, is demonstrated originating from the hybrid polariton state

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis

Abstract: Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis

A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine ‘nonmodel’ and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip conceptThe following funding is acknowledged: Agence Nationale de la Recherche (contract No. ANR-11-LABX- 0057_MITOCROSS to Claude Sauter, Bernard Lorber; contract No. ANR-10-LABX-0036_NETRN to Claude Sauter, Bernard Lorber; contract No. ANR-13-BS07-0007-01 to Eric Girard, Sylvain Engilberge); Ministère des Affaires Etrangères (contract No. PROCOPE Hubert Curien to Claude Sauter, Mario Mörl); Deutsche Forschungsgemeinschaft (contract No. Mo 634/10-1 to Mario Mörl, Heike Betat); Université de Strasbourg [grant No. Initiative d’excellence (IDEX) to Claude Sauter, Raphaël de Wijn]; Centre National de la Recherche Scientifique (grant No. MRCT- 2012_PTI_UPR9002 to Claude Sauter)

SLAIN2 links microtubule plus end–tracking proteins and controls microtubule growth in interphase

SLAIN2’s interactions with multiple different microtubule plus end–tracking proteins stimulate processive microtubule polymerization and ensure proper microtubule organization

Caenorhabditis elegans N-glycan Core β-galactoside Confers Sensitivity towards Nematotoxic Fungal Galectin CGL2

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galβ1,4Fucα1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galβ1,4Fucα1,6GlcNAc trisaccharide at 1.5 Å resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms

Etude structurale du complexe entre la protéine SBP2 et l'ARN SECIS (deux partenaires cruciaux pour la synthèse des sélénoprotéines chez les eucaryotes)

Longtemps considéré comme toxique, le sélénium est aujourd'hui reconnu comme essentiel. Admise comme le 21ème acide aminé, la sélénocystéine (acide aminé U ou Sec) représente la forme biologique majeure du sélénium et constitue une exception à la règle universelle du code génétique. En effet, le codon spécifique de la Sec est UGA, habituellement lu comme signal de terminaison de la traduction. Les sélénoprotéines ainsi produites sont des enzymes impliqués majoritairement dans des processus d'oxydo-réduction et de défense contre les radicaux libres. Elles ont aussi été découvertes impliquées dans un grand nombre de pathologies telles que certaines myopathies, le cancer ou l'infertilité masculine.La première partie de ce travail de thèse a porté sur l'étude de deux partenaires de l'incorporation de la sélénocystéine dans les protéines. Chez les eucaryotes, la protéine SBP2 et l'ARN SECIS (tige-boucle de l'ARNm située dans la région 3' non traduite) font partie d'une machinerie complexe permettant la reprogrammation du codon UGA Sec. En vue de la cristallisation de ce complexe, différentes constructions de la protéine et de l'ARN ont été utilisées. Des protocoles d'expression et de purification permettant d'obtenir les macromolécules en quantité ont été mis au point. Malgré le criblage de plusieurs milliers de conditions, il n'a pas été possible d'obtenir de cristaux, ni de complexe, ni de la protéine seule. La caractérisation biophysique de la protéine SBP2 par diffusion de lumière, ultracentrifugation analytique et RMN a révélé une absence de structuration. Dans les mécanismes moléculaires permettant la synthèse des sélénoprotéines, le complexe SBP2/ARN SECIS agit comme une plateforme pour le recrutement d'autres facteurs qui pourraient être nécessaires à la stabilisation de la protéine SBP2. Certains n'ont pas encore été identifiés. Une autre hypothèse serait que la destructuration de SBP2 soit nécessaire pour son activité biologique comme cela est le cas d'autres protéines intrinsèquement non structurées.La deuxième partie, complètement indépendante, a consisté à étudier les dommages causés par les rayons X sur un cristal de macromolécule biologique pendant une collecte de données cristallographiques. Les modifications structurales engendrées peuvent parfois rendre le phasage et donc la résolution de la structure impossibles. Le suivi de ces dommages sur un cristal d'ARN bromé par l'enregistrement de spectres de fluorescence X au cours d'une collecte de données a été le principal objectif de ce travail. Nous avons observé une modification systématique des spectres de fluorescence se corrélant bien avec la dose cumulée de rayons X provoquant la coupure de la liaison C-Br. Une conséquence pratique est que, par une simple différence entre un spectre quelconque et celui mesuré sur une solution de NaBr (100 % de brome libre), on obtient une excellente estimation du pourcentage de brome encore lié. Ces mesures supplémentaires pourraient permettre d'ajuster l'intensité du faisceau et d'évaluer l'occupation restante à tout moment au cours de la collecte, ceci afin d'améliorer le phasage par la méthode RIP (Radiation-damage-Induced Phasing).Selenium, long considered as a potent toxic substance, is actually necessary for the function of all cells in (probably) all living organisms. Selenocysteine (Sec, U), now widely considered as the 21st amino acid, is the major biological form of selenium. This particular amino acid is specifically incorporated into selenoproteins through a dedicated translation machinery that constitutes an exception to the universal genetic code. Unlike other amino acids, its insertion into proteins (named selenoproteins) requires a translational recoding event that occurs at select UGA codons, which usually signal translation termination. There are 25 selenoproteins identified in human. Whereas most of them have not been attributed a function yet, their implications in a large number of pathologies such as myopathies, cancer or in fertility have been demonstrated.This work focused on the understanding of this atypical incorporation of selenocysteine into proteins. In eukaryotes, SBP2 protein and SECIS RNA (a stem-loop structure in the mRNA located in the 3'-UTR) are two essential factors that allow reprogrammation of the UGA stop codon. In order to visualize their interactions, we have tried to cristallize the complex by using various SECIS RNA sequences and different truncated forms of the SBP2 protein. Expression and purification protocols have been set up and reasonable amounts of macromolecules have been obtained. Besides the screening of thousands of conditions, no crystal has grown. Biophysical characterization by diffusion light scattering, analytical ultracentrifugation and 1D NMR have revealed a lack of structuration of the protein. In fact, in order to allow the synthesis of selenoproteins, SBP2/SECIS RNA complex would act as a platform to recruit other factors that might be needed for the stabilization of SBP2. Some of those factors are still to be discovered. Another hypothesis would be that the non-folded domains of SBP2 are required for its biological function as it is the case for other intrinsically unstructured proteins.The second part of this thesis work, completely independent, aimed at studying the damage to cryo-cooled macromolecular crystals caused by intense X-ray flux. Radiation damage events that affect anomalous scatterers can actually prevent structure determination. We have used fluorescence spectra to monitor radiation damage during data collection on brominated RNA crystals. This type of spectrum is the one usually recorded before a MAD experiment. Collected consecutively or during data collection at regular intervals, we observed modifications of the spectra that are correlated with C-Br bonds cleavage caused by X-rays. They also yielded a good estimate of bromine occupancy over the time of the experiment. These results will be useful to optimize what is now known as the RIP method (Radiation-damage-Induced Phasing) exploiting both anomalous signal of an anomalous scatterer and the 'isomorphous' signal resulting from the X-ray damage.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF