RESUMEN
The chromatophores in Paulinella are evolutionary-early-stage photosynthetic organelles. Biological processes in chromatophores depend on a combination of chromatophore and nucleus-encoded proteins. Interestingly, besides proteins carrying chromatophore-targeting signals, a large arsenal of short chromatophore-targeted proteins (sCTPs; <90 amino acids) without recognizable targeting signals were found in chromatophores. This situation resembles endosymbionts in plants and insects that are manipulated by host-derived antimicrobial peptides. Previously, we identified an expanded family of sCTPs of unknown function, named here "DNA-binding (DB)-sCTPs". DB-sCTPs contain a ~45 amino acid motif that is conserved in some bacterial proteins with predicted functions in DNA processing. Here, we explored antimicrobial activity, DNA-binding capacity, and structures of three purified recombinant DB-sCTPs. All three proteins exhibited antimicrobial activity against bacteria involving membrane permeabilization, and bound to bacterial lipids in vitro. A combination of in vitro assays demonstrated binding of recombinant DB-sCTPs to chromatophore-derived genomic DNA sequences with an affinity in the low nM range. Additionally, we report the 1.2 Å crystal structure of one DB-sCTP. In silico docking studies suggest that helix α2 inserts into the DNA major grove and the exposed residues, that are highly variable between different DB-sCTPs, confer interaction with the DNA bases. Identification of photosystem II subunit CP43 as a potential interaction partner of one DB-sCTP, suggests DB-sCTPs to be involved in more complex regulatory mechanisms. We hypothesize that membrane binding of DB-sCTPs is related to their import into chromatophores. Once inside, they interact with the chromatophore genome potentially providing nuclear control over genetic information processing.
Asunto(s)
Antiinfecciosos , Cromatóforos , Rhizaria , Evolución Biológica , Fotosíntesis/genética , Cromatóforos/metabolismo , Antiinfecciosos/metabolismoRESUMEN
Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5â Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.
Asunto(s)
Isomerasas Aldosa-Cetosa , Fosfomicina , Complejos Multienzimáticos , Toxoplasma , Toxoplasma/enzimología , Toxoplasma/efectos de los fármacos , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/metabolismo , Isomerasas Aldosa-Cetosa/genética , Fosfomicina/farmacología , Fosfomicina/análogos & derivados , Fosfomicina/química , Cristalografía por Rayos X , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , NADP/metabolismo , NADP/química , Humanos , Modelos Moleculares , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Oxidorreductasas/metabolismoRESUMEN
Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus-approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy.
Asunto(s)
Receptores Artificiales , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Palivizumab/farmacología , Palivizumab/uso terapéutico , Receptores Artificiales/metabolismo , Receptores Artificiales/uso terapéutico , Receptores de Citocinas , Citocinas , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Ligandos , Antivirales/farmacología , Antivirales/uso terapéuticoRESUMEN
The liver is beyond any doubt the most important metabolic organ of the human body. This function requires an intensive crosstalk within liver cellular structures, but also with other organs. Membrane transport proteins are therefore of upmost importance as they represent the sensors and mediators that shuttle signals from outside to the inside of liver cells and/or vice versa. In this review, we summarize the known literature of liver transport proteins with a clear emphasis on functional and structural information on ATP binding cassette (ABC) transporters, which are expressed in the human liver. These primary active membrane transporters form one of the largest families of membrane proteins. In the liver, they play an essential role in for example bile formation or xenobiotic export. Our review provides a state of the art and comprehensive summary of the current knowledge of hepatobiliary ABC transporters. Clearly, our knowledge has improved with a breath-taking speed over the last few years and will expand further. Thus, this review will provide the status quo and will lay the foundation for new and exciting avenues in liver membrane transporter research.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Sistema Biliar/metabolismo , Hígado/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Humanos , Simulación de Dinámica Molecular , Relación Estructura-ActividadRESUMEN
Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. KEY POINTS: ⢠An improvement in protein secretion via the use of T1SS ⢠Reaching almost 400 mg/L of soluble lipase into the supernatant ⢠A step forward to making E. coli cells more competitive for applying as a secretion host.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Sistemas de Secreción Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Lipasa/genética , Lipasa/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Ectoine and its derivative hydroxyectoine are widely synthesized or imported by bacteria to fend off the detrimental effects of high osmolarity on cellular hydration and growth. Genes that are connected to a particular physiological process are often found in the same genomic context. We exploited this feature in a comprehensive bioinformatical analysis of 1103 ectoine biosynthetic gene clusters from Bacteria and Archaea through which we identified 415 ect operons that colocalize with genes encoding potential osmolyte transporters. These belong to various importer families. Focusing on the complex ect gene clusters of the alpha-proteobacteria Hyphomonas neptunium and Novoshingobium sp. LH128, we analysed several transporters with respect to their substrate specificities through physiological, molecular and modelling approaches. Accordingly, we identified an MFS-type uptake system specific for ectoines (EctU) and a novel SSS-type ectoine/hydroxyectoine importer (EctI) with a broader substrate profile for osmostress protectants. Furthermore, some ect gene clusters encode a MscS/YbdG-type mechanosensitive channel protein, whose functionality was assessed through down-shock assays. Moreover, our analysis identified the gene for the first putative ectoine/hydroxyectoine-specific efflux system (EctX), a member of the MFS superfamily. Our findings make substantial contributions to the understanding of the ecophysiology of ectoines, key players in microbial osmostress adjustment systems.
Asunto(s)
Aminoácidos Diaminos , Proteínas Bacterianas , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Aminoácidos Diaminos/genética , Aminoácidos Diaminos/metabolismo , Familia de Multigenes , Proteínas de Transporte de Membrana/genética , Archaea/genética , Bacterias/genéticaRESUMEN
Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Proteínas Hemolisinas , Ingeniería de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Regiones Terminadoras Genéticas , Sistemas de Secreción Tipo I/metabolismoRESUMEN
Secretion systems are essential for Gram-negative bacteria, as these nanomachineries allow communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type I secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli, which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC, and the substrate HlyA, a member of the family of repeats in toxins (RTX) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression [T7 expression system, BL21(DE3)-BD]. The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by superresolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS clusters at the outer membrane, generating domains of so-far-undescribed identity. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide, representing a global burden to the health care system. UPEC strains secrete many virulence factors, among these, the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore, and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the superresolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.
Asunto(s)
Proteínas de Escherichia coli , Proteínas Hemolisinas , Escherichia coli Uropatógena , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Sistemas de Secreción Tipo IRESUMEN
Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR2 E2 , with efficient native transaminase (kcat : 69.49±1.77â min-1 ) and artificial esterase (kcat : 3908-0.41â min-1 ) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR2 E2 (pHopt : 8.0-9.5; Topt : 60-65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the ß-keto ester into the ß-keto acid at the hydrolytic site and subsequently into the ß-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR2 E2 PluriZyme was proven with a set of ß-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.
Asunto(s)
Aminoácidos , Transaminasas , Catálisis , Esterasas , Ésteres/química , HidrólisisRESUMEN
ABCB4, also called multidrug-resistant protein 3 (MDR3), is an ATP binding cassette transporter located in the canalicular membrane of hepatocytes that specifically translocates phosphatidylcholine (PC) lipids from the cytoplasmic to the extracellular leaflet. Due to the harsh detergent effect of bile acids, PC lipids provided by ABCB4 are extracted into the bile. While it is well known that bile acids are the major extractor of PC lipids from the membrane into bile, it is unknown whether only PC lipid extraction is improved or whether bile acids also have a direct effect on ABCB4. Using in vitro experiments, we investigated the modulation of ATP hydrolysis of ABC by different bile acids commonly present in humans. We demonstrated that all tested bile acids stimulated ATPase activity except for taurolithocholic acid, which inhibited ATPase activity due to its hydrophobic nature. Additionally, we observed a nearly linear correlation between the critical micelle concentration and maximal stimulation by each bile acid, and that this modulation was maintained in the presence of PC lipids. This study revealed a large effect of 24-nor-ursodeoxycholic acid, suggesting a distinct mode of regulation of ATPase activity compared with other bile acids. In addition, it sheds light on the molecular cross talk of canalicular ABC transporters of the human liver.
Asunto(s)
Ácidos y Sales BiliaresRESUMEN
Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.
Asunto(s)
Acetiltransferasas/química , Aminoácidos Diaminos/biosíntesis , Proteínas Bacterianas/química , Dominio Catalítico , Paenibacillus/química , Cristalografía por Rayos X , Dimerización , Mutagénesis Sitio-Dirigida , Relación Estructura-ActividadRESUMEN
Evolutionary engineering is a powerful method to improve the performance of microbial cell factories, but can typically not be applied to enhance the production of chemicals due to the lack of an appropriate selection regime. We report here on a new strategy based on transcription factor-based biosensors, which directly couple production to growth. The growth of Corynebacterium glutamicum was coupled to the intracellular concentration of branched-chain amino acids, by integrating a synthetic circuit based on the Lrp biosensor upstream of two growth-regulating genes, pfkA and hisD. Modelling and experimental data highlight spatial separation as key strategy to limit the selection of 'cheater' strains that escaped the evolutionary pressure. This approach facilitated the isolation of strains featuring specific causal mutations enhancing amino acid production. We envision that this strategy can be applied with the plethora of known biosensors in various microbes, unlocking evolution as a feasible strategy to improve production of chemicals.
Asunto(s)
Técnicas Biosensibles , Corynebacterium glutamicum , Aminoácidos , Corynebacterium glutamicum/genética , Ingeniería Metabólica , MutaciónRESUMEN
Relaxases of the MOBH family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOBH family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn2+ or Co2+ dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3' of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site.
Asunto(s)
Proteínas Bacterianas/genética , ADN Helicasas/genética , ADN Nucleotidiltransferasas/genética , ADN Bacteriano/genética , Islas Genómicas/genética , Neisseria gonorrhoeae/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Dominio Catalítico , División del ADN , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Nucleotidiltransferasas/química , ADN Nucleotidiltransferasas/metabolismo , ADN Bacteriano/metabolismo , Metales/química , Metales/metabolismo , Neisseria gonorrhoeae/metabolismo , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Ácido NucleicoRESUMEN
ABCB4/MDR3 is located in the canalicular membrane of hepatocytes and translocates PC-lipids from the cytoplasmic to the extracellular leaflet. ABCB4 is an ATP-dependent transporter that reduces the harsh detergent effect of the bile salts by counteracting self-digestion. To do so, ABCB4 provides PC lipids for extraction into bile. PC lipids account for 40% of the entire pool of lipids in the canalicular membrane with an unknown distribution over both leaflets. Extracted PC lipids end up in so-called mixed micelles. Mixed micelles are composed of phospholipids, bile salts, and cholesterol. Ninety to ninety-five percent of the phospholipids are members of the PC family, but only a subset of mainly 16.0-18:1 PC and 16:0-18:2 PC variants are present. To elucidate whether ABCB4 is the key discriminator in this enrichment of specific PC lipids, we used in vitro studies to identify crucial determinants in substrate selection. We demonstrate that PC-lipid moieties alone are insufficient for stimulating ABCB4 ATPase activity, and that at least two acyl chains and the backbone itself are required for a productive interaction. The nature of the fatty acids, like length or saturation has a quantitative impact on the ATPase activity. Our data demonstrate a two-step enrichment and protective function of ABCB4 to mitigate the harsh detergent effect of the bile salts, because ABCB4 can translocate more than just the PC-lipid variants found in bile.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/farmacología , Fosfatidilcolinas/metabolismo , Colesterol/metabolismo , Células HEK293 , HumanosRESUMEN
Ectoine and its derivative 5-hydroxyectoine are compatible solutes and chemical chaperones widely synthesized by Bacteria and some Archaea as cytoprotectants during osmotic stress and high- or low-growth temperature extremes. The function-preserving attributes of ectoines led to numerous biotechnological and biomedical applications and fostered the development of an industrial scale production process. Synthesis of ectoines requires the expenditure of considerable energetic and biosynthetic resources. Hence, microorganisms have developed ways to exploit ectoines as nutrients when they are no longer needed as stress protectants. Here, we summarize our current knowledge on the phylogenomic distribution of ectoine producing and consuming microorganisms. We emphasize the structural enzymology of the pathways underlying ectoine biosynthesis and consumption, an understanding that has been achieved only recently. The synthesis and degradation pathways critically differ in the isomeric form of the key metabolite N-acetyldiaminobutyric acid (ADABA). γ-ADABA serves as preferred substrate for the ectoine synthase, while the α-ADABA isomer is produced by the ectoine hydrolase as an intermediate in catabolism. It can serve as internal inducer for the genetic control of ectoine catabolic genes via the GabR/MocR-type regulator EnuR. Our review highlights the importance of structural enzymology to inspire the mechanistic understanding of metabolic networks at the biological scale.
Asunto(s)
Aminoácidos Diaminos/metabolismo , Bacterias/metabolismo , Hidroliasas/metabolismo , Chaperonas Moleculares/metabolismo , Nutrientes/metabolismo , Aminoácidos Diaminos/química , Hidroliasas/química , Chaperonas Moleculares/química , Estructura Molecular , Nutrientes/química , Presión OsmóticaRESUMEN
Guanylate-binding proteins (GBPs) constitute a family of interferon-inducible guanosine triphosphatases (GTPases) that are key players in host defense against intracellular pathogens ranging from protozoa to bacteria and viruses. So far, human GBP1 and GBP5 as well as murine GBP2 (mGBP2) have been biochemically characterized in detail. Here, with murine GBP7 (mGBP7), a GBP family member with an unconventional and elongated C-terminus is analyzed. The present study demonstrates that mGBP7 exhibits a concentration-dependent GTPase activity and an apparent GTP turnover number of 20â min-1. In addition, fluorescence spectroscopy analyses reveal that mGBP7 binds GTP with high affinity (KD = 0.22â µM) and GTPase activity assays indicate that mGBP7 hydrolyzes GTP to GDP and GMP. The mGBP7 GTPase activity is inhibited by incubation with γ-phosphate analogs and a K51A mutation interfering with GTP binding. SEC-MALS analyses give evidence that mGBP7 forms transient dimers and that this oligomerization pattern is not influenced by the presence of nucleotides. Moreover, a structural model for mGBP7 is provided by homology modeling, which shows that the GTPase possesses an elongated C-terminal (CT) tail compared with the CaaX motif-containing mGBP2 and human GBP1. Molecular dynamics simulations indicate that this tail has transmembrane characteristics and, interestingly, confocal microscopy analyses reveal that the CT tail is required for recruitment of mGBP7 to the parasitophorous vacuole of Toxoplasma gondii.
Asunto(s)
Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas de Unión al GTP/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Ratones , Simulación de Dinámica Molecular , Dominios Proteicos , Toxoplasma/fisiología , Toxoplasmosis/enzimología , Toxoplasmosis/genética , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitologíaRESUMEN
The cofactor-less dioxygenase AqdC of Mycobacteroides abscessus catalyzes the cleavage and thus inactivation of the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone), which plays a central role in the regulation of virulence factor production by Pseudomonas aeruginosa. We present here the crystal structures of AqdC in its native state and in complex with the PQS cleavage product N-octanoylanthranilic acid, and of mutant AqdC proteins in complex with PQS. AqdC possesses an α/ß-hydrolase fold core domain with additional helices forming a cap domain. The protein is traversed by a bipartite tunnel, with a funnel-like entry section leading to an elliptical substrate cavity where PQS positioning is mediated by a combination of hydrophobic interactions and hydrogen bonds, with the substrate's C4 carbonyl and C3 hydroxyl groups tethered by His97 and the catalytic His246, respectively. The side chain of the AqdC-bound product extends deeper into the "alkyl tail section" of the tunnel than PQS, tentatively suggesting product exit via this part of the tunnel. AqdC prefers PQS over congeners with shorter alkyl substituents at C2. Kinetic data confirmed the strict requirement of the active-site base His246 for catalysis, and suggested that evolution of the canonical nucleophile/His/Asp catalytic triad of the hydrolases to an Ala/His/Asp triad is favorable for catalyzing dioxygenolytic PQS ring cleavage.
Asunto(s)
Dioxigenasas/química , Hidrolasas/química , Pseudomonas aeruginosa/metabolismo , Quinolonas/química , Cristalografía por Rayos X , Dioxigenasas/genética , Dioxigenasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Enlace de Hidrógeno , Hidrolasas/genética , Hidrolasas/metabolismo , Cinética , Modelos Moleculares , Mutación , Mycobacterium/enzimología , Mycobacterium/genética , Filogenia , Unión Proteica , Conformación Proteica , Pseudomonas aeruginosa/genética , Quinolonas/metabolismo , Especificidad por SustratoRESUMEN
The rising existence of antimicrobial resistance, confirms the urgent need for new antimicrobial compounds. Lantibiotics are active in a low nanomolar range and represent good compound candidates. The lantibiotic nisin is well studied, thus it is a perfect origin for exploring novel lantibiotics via mutagenesis studies. However, some human pathogens like Streptococcus agalactiae COH1 already express resistance proteins against lantibiotics like nisin. This study presents three nisin variants with mutations in the hinge-region and determine their influence on both the growth inhibition as well as the pore-forming activity. Furthermore, we analyzed the effect of these mutants on the nisin immunity proteins NisI and NisFEG from Lactococcus lactis, as well as the nisin resistance proteins SaNSR and SaNsrFP from Streptococcus agalactiae COH1. We identified the nisin variant 20NMKIV24 with an extended hinge-region, to be an excellent candidate for further studies to eventually overcome the lantibiotic resistance in human pathogens, since these proteins do not recognize this variant well.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Lactococcus lactis/genética , Lipoproteínas/genética , Proteínas de la Membrana/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Variación Genética/genética , Lactococcus lactis/inmunología , Lactococcus lactis/metabolismo , Lipoproteínas/inmunología , Lipoproteínas/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismoRESUMEN
Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Farmacorresistencia Bacteriana/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Streptococcus agalactiae/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Streptococcus agalactiae/química , Relación Estructura-ActividadRESUMEN
The need for new antibiotic compounds is rising and antimicrobial peptides are excellent candidates to fulfill this object. The bacteriocin subgroup lantibiotics, for example, are active in the nanomolar range and target the membranes of mainly Gram-positive bacteria. They bind to lipid II, inhibit cell growth and in some cases form pores within the bacterial membrane, inducing rapid cell death. Pharmaceutical usage of lantibiotics is however hampered by the presence of gene clusters in human pathogenic strains which, when expressed, confer resistance. The human pathogen Streptococcus agalactiae COH1, expresses several lantibiotic resistance proteins resulting in resistance against for example nisin. This study presents a highly potent, pore forming nisin variant as an alternative lantibiotic which bypasses the SaNSR protein. It is shown that this nisin derivate nisinC28P keeps its nanomolar antibacterial activity against L. lactis NZ9000 cells but is not recognized by the nisin resistance protein SaNSR. NisinC28P is cleaved by SaNSR in vitro with a highly decreased efficiency, as shown by an cleavage assay. Furthermore, we show that nisinC28P is still able to form pores in the membranes of L. lactis and is three times more efficient against SaNSR-expressing L. lactis cells than wildtype nisin.