The high resolution structure of tyrocidine A reveals an amphipathic dimer

AbstractTyrocidine A, one of the first antibiotics ever to be discovered, is a cyclic decapeptide that binds to membranes of target bacteria, disrupting their integrity. It is active against a broad spectrum of Gram-positive organisms, and has recently engendered interest as a potential scaffold for the development of new drugs to combat antibiotic-resistant pathogens. We present here the X-ray crystal structure of tyrocidine A at a resolution of 0.95Å. The structure reveals that tyrocidine forms an intimate and highly amphipathic homodimer made up of four beta strands that associate into a single, highly curved antiparallel beta sheet. We used surface plasmon resonance and potassium efflux assays to demonstrate that tyrocidine binds tightly to mimetics of bacterial membranes with an apparent dissociation constant (KD) of 10μM, and efficiently permeabilizes bacterial cells at concentrations equal to and below the KD. Using variant forms of tyrocidine in which the fluorescent probe p-cyano-phenylalanine had been inserted on either the polar or apolar face of the molecule, we performed fluorescence quenching experiments, using both water-soluble and membrane-embedded quenchers. The quenching results, together with the structure, strongly support a membrane association model in which the convex, apolar face of tyrocidine's beta sheet is oriented toward the membrane interior, while the concave, polar face is presented to the aqueous phase

New insights on the mechanism of quinoline-based DNA methyltransferase inhibitors

Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-L-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate

Alpha-amylases de mammifères et d'insectes, relation structure/fonction

AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

-AMYLASES DE MAMMIFERES ET D'INSECTES RELATION STRUCTURE/FONCTION

LES -AMYLASES (-1,4-GLUCAN-4-GLUCANOHYDROLASE, EC 3.2.1.1) CATALYSENT L'HYDROLYSE DES LIAISONS -(1,4) GLYCOSIDIQUES DES COMPOSES DE L'AMIDON. DANS CETTE THESE JE PRESENTE L'ANALYSE STRUCTURALE DE DEUX CLASSES D'ENZYMES, LES -AMYLASES DE MAMMIFERES ET D'INSECTES. MALGRE L'ABONDANCE DE DONNEES BIOCHIMIQUES SUR CES COMPLEXES, LES COMPLEXES ENTRE L'-AMYLASE HUMAINE ET DES OLIGOSACCHARIDES AVAIENT ECHAPPE A TOUTE CARACTERISATION STRUCTURALE. DANS CETTE ETUDE LES STRUCTURES CRISTALLOGRAPHIQUES DE L'-AMYLASE PANCREATIQUE HUMAINE (HPA) EN COMPLEXE AVEC DES INHIBITEURS NATURELS ONT ETE RESOLUES. LE TETRASACCHARIDE ACARBOSE ET UN PSEUDO-PENTASCCHARIDE APPARTENANT A LA FAMILLE DES TRESTATINES ONT MONTRE DES DENSITES ELECTRONIQUES CONTINUES SIMILAIRES CORRESPONDANT A UN PENTASACCHARIDE, OCCUPANT LES SOUS-SITES 3 A +2 DE L'ENZYME, ET PROBABLEMENT RESULTANT D'UNE TRANSGLYCOSYLATION. LA FIXATION DU CUR ACARVIOSINE LIE A UNE UNITE GLUCOSE AUX SOUS-SITES 1 A +2 APPARAIT ETRE UNE ETAPE CRITIQUE DU PROCESSUS D'INTERACTION ENTRE LES -AMYLASES ET LES INHIBITEURS DERIVES DES TRESTATINES. DEUX FORMES CRISTALLINES DU COMPLEXE ENTRE HPA ET UN INHIBITEUR PROTEIQUE (-AI) ISSU DU GRAIN DE HARICOT PHASEOLUS VULGARIS, OBTENUES A DIFFERENTS PH, ONT ETE RESOLUES. LA BOUCLE FLEXIBLE, TYPIQUE DES -AMYLASES DE MAMMIFERES, MONTRE DEUX CONFORMATIONS DIFFERENTES, SUGGERANT UNE SENSIBILITE DE CETTE BOUCLE AU PH. UNE INFORMATION STRUCTURALE EST PROCUREE POUR LE RESIDU ARG 74 QUI N'ETAIT PAS VISIBLE DANS LES PRECEDENTES ANALYSES STRUCTURALES. L'-AMYLASE DE LA LARVE DE TENEBRIO MOLITOR (TMA) A ETE CRISTALLISEE EN COMPLEXE AVEC L'INHIBITEUR -AI. TMA PRESENTE LA STRUCTURE COMMUNE AUX -AMYLASES, DE GRANDES DIFFERENCES AVEC LES -AMYLASES DE MAMMIFERES SE PRODUISANT DANS LES BOUCLES. MALGRE CES DIFFERENCES DANS LES BOUCLES IMPLIQUEES DANS L'INTERACTION, L'INHIBITEUR DU HARICOT EST CAPABLE D'INHIBER A LA FOIS LES -AMYLASES DE MAMMIFERES ET D'INSECTES. LA BOUCLE FLEXIBLE DES -AMYLASES DE MAMMIFERES, TRONQUEE DANS L'-AMYLASE D'INSECTE MONTRE UNE CONFORMATION DIFFERENTE DANS LES STRUCTURES DE TMA NATIVE ET DE TMA EN COMPLEXE AVEC -AI. LA MODELISATION DE L'ATTACHEMENT DES ISOFORMES DE -AI DANS LE SITE ACTIF DE DIFFERENTES -AMYLASES JETTE UN REGARD SUR LA SPECIFICITE DE CES INHIBITEURS.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Thiazotropsin aggregation and its relationship to molecular recognition in the DNA minor groove

Aggregated states have been alluded to for many DNA minor groove binders but details of the molecule-on-molecule relationship have either been under-reported or ignored. Here we report our findings from ITC and NMR measurements carried out with AIK-18/51, a compound representative of the thiazotropsin class of DNA minor groove binders. The free aqueous form of AIK-18/51 is compared with that found in its complex with cognate DNA duplex d(CGACTAGTCG)2. Molecular self-association of AIK-18/51 is consistent with anti-parallel, face-to-face dimer formation, the building block on which the molecule aggregates. This underlying structure is closely allied to the form found in the ligand’s DNA complex. NMR chemical shift and diffusion measurements yield a self-association constant Kass = (61 ± 19) × 103 M- 1 for AIK-18/51 that fits with a stepwise self-assembly model and is consistent with ITC data. The deconstructed energetics of this assembly process are reported with respect to a design strategy for ligand/DNA recognition

A plant-seed inhibitor of two classes of α-amylases: X-ray analysis of Tenebrio molitor larvae α-amylase in complex with the bean Phaseolus vulgaris inhibitor

International audienc