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The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing a degenerate central region. Toward defining mechanistic principles of affinity regulated by degeneracy, we observed an unusual dependence of the affinity of RelA on the identity of the central base pair, which appears to be noncontacted in the complex crystal structures. The affinity of κB sites with A or T at the central position is ~10-fold higher than with G or C. The crystal structures of neither the complexes nor the free κB DNAs could explain the differences in affinity. Interestingly, differential dynamics of several residues were revealed in molecular dynamics simulation studies, where simulation replicates totaling 148 µs were performed on NF-κB:DNA complexes and free κB DNAs. Notably, Arg187 and Arg124 exhibited selectivity in transient interactions that orchestrated a complex interplay among several DNA-interacting residues in the central region. Binding and simulation studies with mutants supported these observations of transient interactions dictating specificity. In combination with published reports, this work provides insights into the nuanced mechanisms governing the discriminatory binding of NF-κB family TFs to κB DNA elements and sheds light on cancer pathogenesis of cRel, a close homolog of RelA.
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ADN , Simulación de Dinámica Molecular , FN-kappa B , Unión Proteica , ADN/metabolismo , Humanos , FN-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/genética , Sitios de Unión , Cristalografía por Rayos XRESUMEN
Covering: 2009 up to August 2023Prenyltransferases (PTs) are involved in the primary and the secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many clinically relevant natural products (NPs). The continued biochemical and structural characterization of the soluble dimethylallyl tryptophan synthase (DMATS) PTs over the past two decades have revealed the significant promise that these enzymes hold as biocatalysts for the chemoenzymatic synthesis of novel drug leads. This is a comprehensive review of DMATSs describing the structure-function relationships that have shaped the mechanistic underpinnings of these enzymes, as well as the application of this knowledge to the engineering of DMATSs. We summarize the key findings and lessons learned from these studies over the past 14 years (2009-2023). In addition, we identify current gaps in our understanding of these fascinating enzymes.
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Dimetilaliltranstransferasa , Dimetilaliltranstransferasa/química , Prenilación , Hongos/metabolismoRESUMEN
Aminoglycosides are bactericidal antibiotics with a broad spectrum of activity, used to treat infections caused mostly by Gram-negative pathogens and as a second-line therapy against tuberculosis. A common resistance mechanism to aminoglycosides is bacterial aminoglycoside acetyltransferase enzymes (AACs), which render aminoglycosides inactive by acetylating their amino groups. In Mycobacterium tuberculosis, an AAC called Eis (enhanced intracellular survival) acetylates kanamycin and amikacin. When upregulated as a result of mutations, Eis causes clinically important aminoglycoside resistance; therefore, Eis inhibitors are attractive as potential aminoglycoside adjuvants for treatment of aminoglycoside-resistant tuberculosis. For over a decade, we have studied Eis and discovered several series of Eis inhibitors. Here, we provide a detailed protocol for a colorimetric assay used for high-throughput discovery of Eis inhibitors, their characterization, and testing their selectivity. We describe protocols for in vitro cell culture assays for testing aminoglycoside adjuvant properties of the inhibitors. A procedure for obtaining crystals of Eis-inhibitor complexes and determining their structures is also presented. Finally, we discuss applicability of these methods to discovery and testing of inhibitors of other AACs.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Proteínas Bacterianas/química , Antibacterianos/farmacología , Aminoglicósidos , Acetiltransferasas/químicaRESUMEN
Novel substituted monohydrazides synthesized for this study displayed broad-spectrum activity against various fungal strains, including a panel of clinically relevant Candida auris strains. The activity of these compounds was either comparable or superior to amphotericin B against most of the fungal strains tested. These compounds possessed fungistatic activity in a time-kill assay and exhibited no mammalian cell toxicity. In addition, they prevented the formation of fungal biofilms. Even after repeated exposures, the Candida albicans ATCC 10231 (strain A) fungal strain did not develop resistance to these monohydrazides.
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Antimicrobial resistance (AMR) poses a significant threat to human health around the world. Though bacterial pathogens can develop resistance through a variety of mechanisms, one of the most prevalent is the production of antibiotic-modifying enzymes like FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. FosB enzymes are found in pathogens such as Staphylococcus aureus, one of the leading pathogens in deaths associated with AMR. fosB gene knockout experiments establish FosB as an attractive drug target, showing that the minimum inhibitory concentration (MIC) of fosfomycin is greatly reduced upon removal of the enzyme. Herein, we have identified eight potential inhibitors of the FosB enzyme from S. aureus by applying high-throughput in silico screening of the ZINC15 database with structural similarity to phosphonoformate, a known FosB inhibitor. In addition, we have obtained crystal structures of FosB complexes to each compound. Furthermore, we have kinetically characterized the compounds with respect to inhibition of FosB. Finally, we have performed synergy assays to determine if any of the new compounds lower the MIC of fosfomycin in S. aureus. Our results will inform future studies on inhibitor design for the FosB enzymes.
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Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA-approved medication for asthma, is a pan-thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor-dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock-treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker-only (CA-125) relapsed ovarian cancer, where the rate of rise of CA-125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well-tolerated therapeutic in the treatment of ovarian cancer.
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Plaquetas , Neoplasias Ováricas , Animales , Femenino , Humanos , Ratones , Plaquetas/metabolismo , Indoles , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Fenilcarbamatos/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The emergence of multidrug-resistant bacteria and the poor efficacy of available antibiotics against these infections have led to the urgent need for novel antibiotics. Acinetobacter baumannii is one of high-priority pathogens due to its ability to mount resistance to different classes of antibiotics. In an effort to provide novel agents in the fight against infections caused by A. baumannii, we synthesized a series of 46 aromatic hydrazides as potential treatments. In this series, 34 compounds were found to be low- to sub-µM inhibitors of A. baumannii growth, with MIC values in the range of 8 µg/mL to ≤0.125 µg/mL against a broad set of multidrug-resistant clinical isolates. These compounds were not highly active against other bacteria. We showed that one of the most potent compounds, 3e, was bacteriostatic and inhibitory to biofilm formation, although it did not disrupt the preformed biofilm. Additionally, we found that these compounds lacked mammalian cytotoxicity. The high antibacterial potency and the lack of mammalian cytotoxicity make these compounds a promising lead series for development of a novel selective anti-A. baumannii antibiotic.
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Acinetobacter baumannii , Animales , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , MamíferosRESUMEN
Over one and a half million people die of tuberculosis (TB) each year. Multidrug-resistant TB infections are especially dangerous, and new drugs are needed to combat them. The high cost and complexity of drug development make repositioning of drugs that are already in clinical use for other indications a potentially time- and money-saving avenue. In this study, we identified among existing drugs five compounds: azelastine, venlafaxine, chloroquine, mefloquine, and proguanil as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis, a causative agent of TB. Eis upregulation is a cause of clinically relevant resistance of TB to kanamycin, which is inactivated by Eis-catalyzed acetylation. Crystal structures of these drugs as well as chlorhexidine in complexes with Eis showed that these inhibitors were bound in the aminoglycoside binding cavity, consistent with their established modes of inhibition with respect to kanamycin. Among three additionally synthesized compounds, a proguanil analogue, designed based on the crystal structure of the Eis-proguanil complex, was 3-fold more potent than proguanil. The crystal structures of these compounds in complexes with Eis explained their inhibitory potencies. These initial efforts in rational drug repositioning can serve as a starting point in further development of Eis inhibitors.
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Acetiltransferasas , Mycobacterium tuberculosis , Tuberculosis , Humanos , Acetiltransferasas/antagonistas & inhibidores , Antituberculosos/farmacología , Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Kanamicina/farmacología , Kanamicina/química , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Proguanil/metabolismo , Tuberculosis/tratamiento farmacológicoRESUMEN
The Gram-positive pathogen Staphylococcus aureus is a leading cause of antimicrobial resistance related deaths worldwide. Like many pathogens with multidrug-resistant strains, S. aureus contains enzymes that confer resistance through antibiotic modification(s). One such enzyme present in S. aureus is FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. fosB gene knockout experiments show that the minimum inhibitory concentration (MIC) of fosfomycin is significantly reduced when the FosB enzyme is not present. This suggests that inhibition of FosB could be an effective method to restore fosfomycin activity. We used high-throughput in silico-based screening to identify small-molecule analogues of fosfomycin that inhibited thiol transferase activity. Phosphonoformate (PPF) was a top hit from our approach. Herein, we have characterized PPF as a competitive inhibitor of FosB from S. aureus (FosBSa) and Bacillus cereus (FosBBc). In addition, we have determined a crystal structure of FosBBc with PPF bound in the active site. Our results will be useful for future structure-based development of FosB inhibitors that can be delivered in combination with fosfomycin in order to increase the efficacy of this antibiotic.
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Fosfomicina , Antibacterianos/química , Foscarnet/metabolismo , Foscarnet/farmacología , Fosfomicina/química , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/metabolismo , Transferasas/metabolismo , Farmacorresistencia Bacteriana , Proteínas Bacterianas/metabolismoRESUMEN
DNA coordinating platinum (Pt) containing compounds cisplatin and carboplatin have been used for the treatment of ovarian cancer therapy for four decades. However, recurrent Pt-resistant cancers are a major cause of mortality. To combat Pt-resistant ovarian cancers, we designed and synthesized a conjugate of an anticancer drug mithramycin with a reactive Pt(II) bearing moiety, which we termed mithplatin. The conjugates displayed both the Mg2+ -dependent noncovalent DNA binding characteristic of mithramycin and the covalent crosslinking to DNA of the Pt. The conjugate was three times as potent as cisplatin against ovarian cancer cells. The DNA lesions caused by the conjugate led to the generation of DNA double-strand breaks, as also observed with cisplatin. Nevertheless, the conjugate was highly active against both Pt-sensitive and Pt-resistant ovarian cancer cells. This study paves the way to developing mithplatins to combat Pt-resistant ovarian cancers.
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Antineoplásicos , Neoplasias Ováricas , Humanos , Femenino , Cisplatino/farmacología , Cisplatino/química , Plicamicina/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , ADN/metabolismo , Línea Celular Tumoral , Resistencia a AntineoplásicosRESUMEN
Bacterial DNA primase DnaG is an attractive target for antibiotic discovery since it plays an essential role in DNA replication. Over the last 10 years, we have developed and optimized a robust colorimetric assay that enabled us to identify and validate inhibitors of bacterial primases. Here, we provide a detailed protocol for this colorimetric assay for DnaG from three different pathogenic bacteria (Mycobacterium tuberculosis, Bacillus anthracis, and Staphylococcus aureus), which can be performed in high throughput. We also describe secondary assays to characterize hits from this high-throughput screening assay. These assays are designed to identify inhibitors of the coupled enzyme inorganic pyrophosphatase, DNA binding agents, and elucidate the mode of inhibition of primase inhibitors.
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ADN Primasa , Mycobacterium tuberculosis , Colorimetría , Bioensayo , ADN BacterianoRESUMEN
A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 â¼ 1 µM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.
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Acetiltransferasas , Mycobacterium tuberculosis , Acetiltransferasas/química , Aminoglicósidos/farmacología , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antituberculosos/química , Proteínas Bacterianas , Bencilaminas/farmacología , Kanamicina/química , Kanamicina/farmacología , Mamíferos/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
Antimicrobial drug resistance is a major health issue plaguing healthcare worldwide and leading to hundreds of thousands of deaths globally each year. Tackling this problem requires discovery and development of new antibacterial agents. In this study, we discovered novel 6-(1-substituted pyrrole-2-yl)-s-triazine containing compounds that potently inhibited the growth of Staphylococcus aureus regardless of its methicillin-resistant status, displaying minimum inhibitory concentration (MIC) values as low as 1 µM. The presence of a single imidazole substituent was critical to the antibacterial activity of these compounds. Some of the compounds also inhibited several nontubercular mycobacteria. We have shown that these molecules are potent bacteriostatic agents and that they are nontoxic to mammalian cells at relevant concentrations. Further development of these compounds as novel antimicrobial agents will be aimed at expanding our armamentarium of antibiotics.
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Antibacterianos , Staphylococcus aureus Resistente a Meticilina , Animales , Antibacterianos/farmacología , Mamíferos , Pruebas de Sensibilidad Microbiana , Pirroles/farmacología , Triazinas/farmacologíaRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a deadly bacterial disease. Drug-resistant strains of Mtb make eradication of TB a daunting task. Overexpression of the enhanced intracellular survival (Eis) protein by Mtb confers resistance to the second-line antibiotic kanamycin (KAN). Eis is an acetyltransferase that acetylates KAN, inactivating its antimicrobial function. Development of Eis inhibitors as KAN adjuvant therapeutics is an attractive path to forestall and overcome KAN resistance. We discovered that an antipsychotic drug, haloperidol (HPD, 1), was a potent Eis inhibitor with IC50 = 0.39 ± 0.08 µM. We determined the crystal structure of the Eis-haloperidol (1) complex, which guided synthesis of 34 analogues. The structure-activity relationship study showed that in addition to haloperidol (1), eight analogues, some of which were smaller than 1, potently inhibited Eis (IC50 ≤ 1 µM). Crystal structures of Eis in complexes with three potent analogues and droperidol (DPD), an antiemetic and antipsychotic, were determined. Three compounds partially restored KAN sensitivity of a KAN-resistant Mtb strain K204 overexpressing Eis. The Eis inhibitors generally did not exhibit cytotoxicity against mammalian cells. All tested compounds were modestly metabolically stable in human liver microsomes, exhibiting 30-60% metabolism over the course of the assay. While direct repurposing of haloperidol as an anti-TB agent is unlikely due to its neurotoxicity, this study reveals potential approaches to modifying this chemical scaffold to minimize toxicity and improve metabolic stability, while preserving potent Eis inhibition.
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Many essential enzymes in bacteria remain promising potential targets of antibacterial agents. In this study, we discovered that dequalinium, a topical antibacterial agent, is an inhibitor of Staphylococcus aureus primase DnaG (SaDnaG) with low-micromolar minimum inhibitory concentrations against several S. aureus strains, including methicillin-resistant bacteria. Mechanistic studies of dequalinium and a series of nine of its synthesized analogues revealed that these compounds are single-stranded DNA bisintercalators that penetrate a bacterium by compromising its membrane. The best compound of this series likely interacts with DnaG directly, inhibits both staphylococcal cell growth and biofilm formation, and displays no significant hemolytic activity or toxicity to mammalian cells. This compound is an excellent lead for further development of a novel anti-staphylococcal therapeutic.
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Antibacterianos/farmacología , ADN Primasa/antagonistas & inhibidores , ADN de Cadena Simple/farmacología , Desarrollo de Medicamentos , Inhibidores Enzimáticos/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Línea Celular , ADN Primasa/metabolismo , ADN de Cadena Simple/síntesis química , ADN de Cadena Simple/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/enzimologíaRESUMEN
Ovarian cancer is a highly deadly malignancy in which recurrence is considered incurable. Resistance to platinum-based chemotherapy bodes a particularly abysmal prognosis, underscoring the need for novel therapeutic agents and strategies. The use of mithramycin, an antineoplastic antibiotic, has been previously limited by its narrow therapeutic window. Recent advances in semisynthetic methods have led to mithramycin analogs with improved pharmacological profiles. Mithramycin inhibits the activity of the transcription factor Sp1, which is closely linked with ovarian tumorigenesis and platinum-resistance. This article summarizes recent clinical developments related to mithramycin and postulates a role for the use of mithramycin, or its analog, in the treatment of platinum-resistant ovarian cancer.
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ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with function of other factors. Mithramycin (MTM) is an anti-cancer, DNA binding natural product that functions as a potent antagonist of ERG and FLI1 by an unknown mechanism. We present a series of crystal structures of the DNA binding domain (DBD) of ERG/FLI1 culminating in a structure of a high-order complex of the ERG/FLI1 DBD, transcription factor Runx2, core-binding factor beta (Cbfß), and MTM on a DNA enhancer site, along with supporting DNA binding studies using MTM and its analogues. Taken together, these data provide insight into allosteric mechanisms underlying ERG and FLI1 transactions and their disruption by MTM analogues.
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Antibióticos Antineoplásicos/farmacología , Plicamicina/farmacología , Proteína Proto-Oncogénica c-fli-1/química , Regulación Alostérica/efectos de los fármacos , Antibióticos Antineoplásicos/química , Sitios de Unión , Subunidad alfa 1 del Factor de Unión al Sitio Principal/química , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/química , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Plicamicina/química , Unión Proteica , Proteína Proto-Oncogénica c-fli-1/metabolismo , Regulador Transcripcional ERG/química , Regulador Transcripcional ERG/metabolismoRESUMEN
Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2'-oxime (MTMox) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTMox analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2'-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTMox32E (a Phe-Trp dipeptide-based 2'-conjugate) for in vivo testing. Relative to MTM, MTMox32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.
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Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/farmacocinética , Neoplasias Óseas/tratamiento farmacológico , Oximas/química , Plicamicina/química , Sarcoma de Ewing/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/química , Apoptosis , Neoplasias Óseas/patología , Proliferación Celular , Femenino , Humanos , Ratones , Ratones SCID , Sarcoma de Ewing/patología , Distribución Tisular , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ETS family transcription factors control development of different cell types in humans, whereas deregulation of these proteins leads to severe developmental syndromes and cancers. One of a few members of the ETS family that are known to act solely as repressors, ERF, is required for normal osteogenesis and hematopoiesis. Another important function of ERF is acting as a tumor suppressor by antagonizing oncogenic fusions involving other ETS family factors. The structure of ERF and the DNA binding properties specific to this protein have not been elucidated. In this study, we determined two crystal structures of the complexes of the DNA binding domain of ERF with DNA. In one, ERF is in a distinct dimeric form, with Cys72 in a reduced state. In the other, two dimers of ERF are assembled into a tetramer that is additionally locked by two Cys72-Cys72 disulfide bonds across the dimers. In the tetramer, the ERF molecules are bound to a pseudocontinuous DNA on the same DNA face at two GGAA binding sites on opposite strands. Sedimentation velocity analysis showed that this tetrameric assembly forms on continuous DNA containing such tandem sites spaced by 7 bp. Our bioinformatic analysis of three previously reported sets of ERF binding loci across entire genomes showed that these loci were enriched in such 7 bp spaced tandem sites. Taken together, these results strongly suggest that the observed tetrameric assembly is a functional state of ERF in the human cell.
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Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.