RESUMEN
Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Bacterianas/metabolismo , Origen de Réplica , Bacterias/genética , ADN , ADN de Cadena Simple/genética , ADN Bacteriano/metabolismo , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismoRESUMEN
Human cystatin C (CysC) is an amyloid forming protein involved in the hereditary cerebral amyloid angiopathy (HCCAA) that affects arteries in the brain and the peripheral nervous system. In this study we measured the influence of several substances on human CysC aggregation and amyloid fibril formation, induced at pH 4 in vitro. The effect of three polyphenols: resveratrol, quercetin and curcumin and of two antioxidants: vitamin C (VitC) and N-acetyl-L-cysteine (NAC) was explored as well as the effect of sulphoraphane (SF) and α-lipoic acid (AL). The formation of amyloid fibrils was followed by Thioflavin T (ThT) fluorescence and by transmission electron microscopy (TEM). Effects on the length of the lag phase were revealed by following the increase of ThT fluorescence intensity with time. The amount and morphology of fibrils in comparison to prefibrillar aggregates and globular oligomers were evaluated by TEM at the plateau stage of the reaction. Thermal stabilization of the CysC monomer by the small compounds was measured by differential scanning fluorimetry (DSF). NAC, VitC and SF exhibited the largest inhibitory effect on amyloid fibril growth. The effects of polyphenols were not significant, apart from resveratrol, which partly inhibited the amyloid fibril growth.
Asunto(s)
Amiloide/química , Antioxidantes/farmacología , Cistatina C/química , Polifenoles/farmacología , Proteínas Recombinantes/química , Dicroismo Circular/métodos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Secundaria de Proteína/efectos de los fármacosRESUMEN
To achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.
Asunto(s)
Acetilglucosaminidasa/metabolismo , Proteínas Bacterianas/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/enzimología , Acetilglucosaminidasa/química , Acetilglucosaminidasa/genética , Regulación Alostérica , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Catálisis , Dominio Catalítico , Hidrólisis , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , N-Acetil Muramoil-L-Alanina Amidasa/química , N-Acetil Muramoil-L-Alanina Amidasa/genética , Dominios Proteicos , Staphylococcus aureus/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Autolysin E (AtlE) is a cell wall degrading enzyme that catalyzes the hydrolysis of the ß-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units of the bacterial peptidoglycan. Using our recently determined crystal structure of AtlE from Staphylococcus aureus and a combination of pharmacophore modeling, similarity search, and molecular docking, a series of (Phenylureido)piperidinyl benzamides were identified as potential binders and surface plasmon resonance (SPR) and saturation-transfer difference (STD) NMR experiments revealed that discovered compounds bind to AtlE in a lower micromolar range. (phenylureido)piperidinyl benzamides are the first reported non-substrate-like compounds that interact with this enzyme and enable further study of the interaction of small molecules with bacterial AtlE as potential inhibitors of this target.
