RESUMEN
Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit ß5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of ß5c/X but not ß5i, thereby explaining the selectivity of PR-957 for ß5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.
Asunto(s)
Cristalografía por Rayos X , Complejo de la Endopetidasa Proteasomal/química , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Alineación de SecuenciaRESUMEN
The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Extensión de la Cadena Peptídica de Translación , Factor 2 de Elongación Peptídica/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalografía por Rayos X , Peptidil-Prolil Isomerasa F , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Resonancia Magnética Nuclear Biomolecular , Factor 2 de Elongación Peptídica/química , Factor 2 de Elongación Peptídica/genética , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-ActividadRESUMEN
Cystargolides and belactosins are natural products with a distinct dipeptide structure and an electrophilic ß-lactone warhead. They are known to inhibit proteases such as the proteasome or caseinolytic protease P, highlighting their potential in treating cancers and neurodegenerative diseases. Recent genetic analyses have shown homology between the biosynthetic pathways of the two inhibitors. Here, we characterize the O-methyltransferases BelI and CysG, which catalyze the initial step of ß-lactone formation. Employing techniques such as crystallography, computational analysis, mutagenesis, and activity assays, we identified a His-His-Asp (HHD) motif in the active sites of the two enzymes, which is crucial for binding a catalytically active calcium ion. Our findings thus elucidate a conserved divalent metal-dependent mechanism in both biosynthetic pathways that distinguishes BelI and CysG from previously characterized O-methyltransferases.
RESUMEN
Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a ß-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin ß-lactone warheads.
Asunto(s)
Dipéptidos , Metiltransferasas , Streptomycetaceae , Vías Biosintéticas , Dipéptidos/metabolismo , Lactonas/metabolismo , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Familia de Multigenes , Streptomyces coelicolor/genética , Streptomycetaceae/enzimología , Streptomycetaceae/genéticaRESUMEN
Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled by several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Reparación del ADN , Proteínas de Unión al ADN/química , ADN/química , Proteínas de Schizosaccharomyces pombe/química , Proteína de la Xerodermia Pigmentosa del Grupo A/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de la radiación , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Línea Celular , Cisplatino/química , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/efectos de la radiación , Formaldehído/química , Células HeLa , Humanos , Cinética , Ratones , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
Photolipids have emerged as attractive tools for the optical control of lipid functions. They often contain an azobenzene photoswitch that imparts a cis double-bond upon irradiation. Herein, we present the application of photoswitching to a lipidated natural product, the potent proteasome inhibitor cepafungin I. Several azobenzene-containing lipids were attached to the cyclopeptide core, yielding photoswitchable derivatives. Most notably, PhotoCep4 exhibited a 10-fold higher cellular potency in its light-induced cis-form, matching the potency of natural cepafungin I. The length of the photolipid tail and distal positioning of the azobenzene photoswitch with respect to the macrocycle is critical for this activity. In a proteome-wide experiment, light-triggered PhotoCep4 modulation showed high overlap with constitutively active cepafungin I. The mode of action was studied using crystallography and revealed an identical binding of the cyclopeptide in comparison to cepafungin I, suggesting that differences in their cellular activity originate from switching the tail structure. The photopharmacological approach described herein could be applicable to many other natural products as lipid conjugation is common and often necessary for potent activity. Such lipids are often introduced late in synthetic routes, enabling facile chemical modifications.
Asunto(s)
Compuestos Azo , Lipopéptidos , Lipopéptidos/farmacología , Proteolisis , Compuestos Azo/química , Péptidos Cíclicos/farmacologíaRESUMEN
The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural ß-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by ß-lactones, the predominant class of ClpP inhibitors.
Asunto(s)
Dipéptidos , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Dominio Catalítico , Dipéptidos/metabolismo , Virulencia , Endopeptidasa Clp/metabolismoRESUMEN
Dithiolopyrrolone (DTP) natural products are produced by several different bacteria and have potent antibacterial, antifungal and anticancer activities. While the amide of their DTP core can be methylated to fine-tune bioactivity, the enzyme responsible for the amide N-methylation has remained elusive in most taxa. Here, we identified the amide methyltransferase XrdM that is responsible for xenorhabdin (XRD) methylation in Xenorhabdus doucetiae but encoded outside of the XRD gene cluster. XrdM turned out to be isofunctional with the recently reported methyltransferase DtpM, that is involved in the biosynthesis of the DTP thiolutin, although its X-ray structure is unrelated to that of DtpM. To investigate the structural basis for ligand binding in both enzymes, we used X-ray crystallography, modeling, site-directed mutagenesis, and kinetic activity assays. Our study expands the limited knowledge of post-non-ribosomal peptide synthetase (NRPS) amide methylation in DTP biosynthesis and reveals an example of convergent evolution of two structurally completely different enzymes for the same reaction in different organisms.
RESUMEN
Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Choque Térmico Pequeñas/química , Chaperonas Moleculares/química , Conformación Proteica , Secuencia de Aminoácidos , Animales , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/clasificación , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/metabolismo , Concentración de Iones de Hidrógeno , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Filogenia , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
The development of novel anti-infectives requires unprecedented strategies targeting pathways which are solely present in pathogens but absent in humans. Following this principle, we developed inhibitors of lipoic acid (LA) salvage, a crucial pathway for the survival of LA auxotrophic bacteria and parasites but non-essential in human cells. An LA-based probe was selectively transferred onto substrate proteins via lipoate protein ligase (LPL) in intact cells, and their binding sites were determined by mass spectrometry. Probe labeling served as a proxy of LPL activity, enabling in situ screenings for cell-permeable LPL inhibitors. Profiling a focused compound library revealed two substrate analogs (LAMe and C3) as inhibitors, which were further validated by binding studies and co-crystallography. Importantly, LAMe exhibited low toxicity in human cells and achieved killing of Plasmodium falciparum in erythrocytes with an EC50 value of 15â µM, making it the most effective LPL inhibitor reported to date.
Asunto(s)
Parásitos , Animales , Humanos , Proteómica , Plasmodium falciparum , Bacterias , EritrocitosRESUMEN
Dioxygenases catalyze stereoselective oxygen atom transfer in metabolic pathways of biological, industrial, and pharmaceutical importance, but their precise chemical principles remain controversial. The α-ketoglutarate (αKG)-dependent dioxygenase AsqJ synthesizes biomedically active quinolone alkaloids via desaturation and subsequent epoxidation of a carbon-carbon bond in the cyclopeptin substrate. Here, we combine high-resolution X-ray crystallography with enzyme engineering, quantum-classical (QM/MM) simulations, and biochemical assays to describe a peroxidic intermediate that bridges the substrate and active site metal ion in AsqJ. Homolytic cleavage of this moiety during substrate epoxidation generates an activated high-valent ferryl (FeIV = O) species that mediates the next catalytic cycle, possibly without the consumption of the metabolically valuable αKG cosubstrate. Our combined findings provide an important understanding of chemical bond activation principles in complex enzymatic reaction networks and molecular mechanisms of dioxygenases.
Asunto(s)
Dioxigenasas , Carbono , Catálisis , Dominio Catalítico , Dioxigenasas/química , Ácidos Cetoglutáricos/metabolismo , Oxígeno/químicaRESUMEN
Understanding antibiotic resistance mechanisms is central to the development of anti-infective therapies and genomics-based drug discovery. Yet, many knowledge gaps remain regarding the resistance strategies employed against novel types of antibiotics from less-explored producers such as anaerobic bacteria, among them the Clostridia. Through the use of genome editing and functional assays, we found that CtaZ confers self-resistance against the copper chelator and gyrase inhibitor closthioamide (CTA) in Ruminiclostridium cellulolyticum. Bioinformatics, biochemical analyses, and X-ray crystallography revealed CtaZ as a founding member of a new group of GyrI-like proteins. CtaZ is unique in binding a polythioamide scaffold in a ligand-optimized hydrophobic pocket, thereby confining CTA. By genome mining using CtaZ as a handle, we discovered previously overlooked homologs encoded by diverse members of the phylum Firmicutes, including many pathogens. In addition to characterizing both a new role for a GyrI-like domain in self-resistance and unprecedented thioamide binding, this work aids in uncovering related drug-resistance mechanisms.
Asunto(s)
Bacterias Anaerobias , Proteínas Portadoras , Antibacterianos/química , Bacterias Anaerobias/genética , Proteínas Portadoras/genética , Farmacorresistencia Microbiana , Edición GénicaRESUMEN
The glidobactin-like natural products (GLNPs) glidobactinâ A and cepafunginâ I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Asunto(s)
Photorhabdus/genética , Inhibidores de Proteasoma/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Diseño de Fármacos , Escherichia coli/metabolismo , Familia de Multigenes/genética , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Photorhabdus/metabolismo , Inhibidores de Proteasoma/química , Relación Estructura-ActividadRESUMEN
Loss of function mutations in the PTEN-induced kinase 1 (PINK1) kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies, we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor and GTPase activating protein. In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly, we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and nuclear magnetic resonance structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential cross-talk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Fosforilación/fisiología , Proteínas Quinasas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Células HEK293 , Humanos , Mitocondrias/metabolismo , Trastornos Parkinsonianos/etiología , Trastornos Parkinsonianos/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMEN
Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.
Asunto(s)
Carbono/metabolismo , Gliotoxina/biosíntesis , Azufre/metabolismo , Aspergillus fumigatus/metabolismo , Carbono/química , Gliotoxina/química , Glutatión/química , Glutatión/metabolismo , Estructura Molecular , Azufre/químicaRESUMEN
Inhibition of the immunoproteasome subunit ß5i alleviates autoimmune diseases in preclinical studies and represents a promising new anti-inflammatory therapy. However, the lack of structural data on the human immunoproteasome still hampers drug design. Here, we systematically determined the potency of seven α' ß' epoxyketone inhibitors with varying N-caps and P3-stereochemistry for mouse/human ß5c/ß5i and found pronounced differences in their subunit and species selectivity. Using X-ray crystallography, the compounds were analyzed for their modes of binding to chimeric yeast proteasomes that incorporate key parts of human ß5c, human ß5i or mouse ß5i and the neighboring ß6 subunit. The structural data reveal exceptional conformations for the most selective human ß5i inhibitors and highlight subtle structural differences as the major reason for the observed species selectivity. Altogether, the presented results validate the humanized yeast proteasome as a powerful tool for structure-based development of ß5i inhibitors with potential clinical applications.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl-CH3 ) to THF in the catabolism of glycine betaine (GB). Despite its lack of sequence identity with known structures, we could show that MtgA forms a homodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of a trapped reaction intermediate (THF-CH3 )+ reveal conformational rearrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl-p-aminobenzoate moiety (THF tail) is unique. Apart from snapshots of individual reaction steps of MtgA, structure-based mutagenesis combined with enzymatic activity assays allowed a mechanistic description of the methyl transfer between Cbl-CH3 and THF. Altogether, the THF-tail-binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF-mediated methyl transfer.
Asunto(s)
Betaína/metabolismo , Desulfitobacterium/química , Tetrahidrofolatos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Betaína/química , Biocatálisis , Cristalografía por Rayos X , Desulfitobacterium/metabolismo , Metilación , Modelos Moleculares , Estructura Molecular , Tetrahidrofolatos/químicaRESUMEN
Acyldepsipeptide (ADEP) is an exploratory antibiotic with a novel mechanism of action. ClpP, the proteolytic core of the caseinolytic protease, is deregulated towards unrestrained proteolysis. Here, we report on the mechanism of ADEP resistance in Firmicutes. This bacterial phylum contains important pathogens that are relevant for potential ADEP therapy. For Staphylococcus aureus, Bacillus subtilis, enterococci and streptococci, spontaneous ADEP-resistant mutants were selected in vitro at a rate of 10-6 . All isolates carried mutations in clpP. All mutated S. aureus ClpP proteins characterised in this study were functionally impaired; this increased our understanding of the mode of operation of ClpP. For molecular insights, crystal structures of S. aureus ClpP bound to ADEP4 were determined. Well-resolved N-terminal domains in the apo structure allow the pore-gating mechanism to be followed. The compilation of mutations presented here indicates residues relevant for ClpP function and suggests that ADEP resistance will occur at a lower rate during the infection process.
Asunto(s)
Antibacterianos/farmacología , Depsipéptidos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Endopeptidasa Clp/antagonistas & inhibidores , Firmicutes/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Depsipéptidos/química , Endopeptidasa Clp/metabolismo , Firmicutes/enzimología , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Mutación , Staphylococcus aureus/enzimologíaRESUMEN
Aryl polyene (APE) pigments are a widely distributed class of bacterial polyketides. So far, little is known about the biosynthesis of these compounds, which are produced by a novel type II polyketide synthase (PKS). We have identified all enzymes involved in APE biosynthesis and determined their peculiar functions. The biosynthesis was reconstituted in vitro, and ACP-bound intermediates were assigned for each reaction step by HPLC-MS. Native mass spectrometry experiments identified four stable complexes: the acyl-carrier proteins ApeE and ApeF bound to the thioesterase ApeK, the dehydratases ApeI and ApeP, and the ketosynthase ApeO in complex with its chain-length factor ApeC. X-ray structures of the heterodimeric ApeO:ApeC and ApeI:ApeP complexes depict striking protein-protein interactions. Altogether, our study elucidated mechanistic aspects of APE biosynthesis that unifies elements of type II fatty acid and PKS systems, but in addition includes novel enzyme complexes.
Asunto(s)
Biocatálisis , Pigmentos Biológicos/biosíntesis , Polienos/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Pigmentos Biológicos/química , Polienos/química , Conformación ProteicaRESUMEN
The biotin-streptavidin interaction is among the strongest known in nature. Herein, the site-directed incorporation of biotin and 2-iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2-Iminobiotin lysine was employed for protein purification based on the pH-dependent dissociation constant to streptavidin. By using the high-affinity binding of biotin lysine, the bacterial protein RecA could be specifically isolated and its interaction partners analyzed. Furthermore, the biotinylation approach was successfully transferred to mammalian cells. Stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin of the different biotin analogues make this approach an attractive tool for protein interaction studies, protein immobilization, and the generation of well-defined protein-drug conjugates.