Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic.

ABSTRACT: DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.

SUBMITTER: Feng L

PROVIDER: S-EPMC7980463 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA.

Project description:The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose beta-strands and turns within the right-handed quadrilateral beta-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.

| S-EPMC2606005 | biostudies-literature

Structural and biochemical analysis of the pentapeptide repeat protein EfsQnr, a potent DNA gyrase inhibitor.

Project description:The chromosomally encoded Qnr homolog protein from Enterococcus faecalis (EfsQnr), when expressed, confers to its host a decreased susceptibility to quinolones and consists mainly of tandem repeats, which is consistent with belonging to the pentapeptide repeat family of proteins (PRPs). EfsQnr was cloned with an N-terminal 6× His tag and purified to homogeneity. EfsQnr partially protected DNA gyrase from fluoroquinolone inhibition at concentrations as low as 20 nM. EfsQnr inhibited the ATP-dependent supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC(50)) of 1.2 ?M, while no significant inhibition of ATP-independent relaxation activity was observed. EfsQnr was cytotoxic when overexpressed in Escherichia coli, resulting in the clumping of cells and a loss of viability. The X-ray crystal structure of EfsQnr was determined to 1.6-Å resolution. EfsQnr exhibits the right-handed quadrilateral beta-helical fold typical of PRPs, with features more analogous to MfpA (mycobacterium fluoroquinolone resistance pentapeptide) than to the PRPs commonly found in cyanobacteria.

| S-EPMC3019687 | biostudies-literature

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes.

Project description:DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.

| S-EPMC7897471 | biostudies-literature

The pentapeptide repeat proteins MfpAMt and QnrB4 exhibit opposite effects on DNA gyrase catalytic reactions and on the ternary gyrase-DNA-quinolone complex.

Project description:MfpA(Mt) and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA(Mt) gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA(Mt) on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 microM, MfpA(Mt) inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 microM. We showed that the D87 residue in GyrA has a major role in the MfpA(Mt)-gyrase interaction, as D87H and D87G substitutions abolished MfpA(Mt) inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpA(Mt) and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 microM as described for other Qnr proteins, but MfpA(Mt) did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA(Mt) also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpA(Mt) and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.

| S-EPMC2648189 | biostudies-literature

Intrinsic resistance of Mycobacterium smegmatis to fluoroquinolones may be influenced by new pentapeptide protein MfpA.

Project description:The fluoroquinolones (FQ) are used in the treatment of Mycobacterium tuberculosis, but the development of resistance could limit their effectiveness. FQ resistance (FQ(R)) is a multistep process involving alterations in the type II topoisomerases and perhaps in the regulation of efflux pumps, but several of the steps remain unidentified. Recombinant plasmid pGADIV was selected from a genomic library of wild-type (WT), FQ-sensitive M. smegmatis by its ability to confer low-level resistance to sparfloxacin (SPX). In WT M. smegmatis, pGADIV increased the MICs of ciprofloxacin (CIP) by fourfold and of SPX by eightfold, and in M. bovis BCG it increased the MICs of both CIP and SPX by fourfold. It had no effect on the accumulation of (14)C-labeled CIP or SPX. The open reading frame responsible for the increase in FQ(R), mfpA, encodes a putative protein belonging to the family of pentapeptides, in which almost every fifth amino acid is either leucine or phenylalanine. Very similar proteins are also present in M. tuberculosis and M. avium. The MICs of CIP and SPX were lower for an M. smegmatis mutant strain lacking an intact mfpA gene than for the WT strain, suggesting that, by some unknown mechanism, the gene product plays a role in determining the innate level of FQ(R) in M. smegmatis.

| S-EPMC90842 | biostudies-literature

Molecular basis for the differential quinolone susceptibility of mycobacterial DNA gyrase.

Project description:DNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind both Escherichia coli and Mycobacterium tuberculosis gyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while in E. coli holoenzyme is the target. The binding of MFX to GyrA of M. tuberculosis correlates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

| S-EPMC4023751 | biostudies-literature

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase.

Project description:ObjectivesTo evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches.MethodsRedx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863.ResultsBoth compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive.ConclusionsRedx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.

| S-EPMC7556816 | biostudies-literature

Benzimidazoles: novel mycobacterial gyrase inhibitors from scaffold morphing.

Project description:Type II topoisomerases are well conserved across the bacterial species, and inhibition of DNA gyrase by fluoroquinolones has provided an attractive option for treatment of tuberculosis (TB). However, the emergence of fluoroquinolone-resistant strains of Mycobacterium tuberculosis (Mtb) poses a threat for its sustainability. A scaffold hopping approach using the binding mode of novel bacterial topoisomerase inhibitors (NBTIs) led to the identification of a novel class of benzimidazoles as DNA gyrase inhibitors with potent anti-TB activity. Docking of benzimidazoles to a NBTI bound crystal structure suggested that this class of compound makes key contacts in the enzyme active site similar to the reported NBTIs. This observation was further confirmed through the measurement of DNA gyrase inhibition, and activity against Mtb strains harboring mutations that confer resistance to aminopiperidines based NBTIs and Mtb strains resistant to moxifloxacin. Structure-activity relationship modification at the C-7 position of the left-hand side ring provided further avenue to improve hERG selectivity for this chemical series that has been the major challenges for NBTIs.

| S-EPMC4094266 | biostudies-literature

DNA-induced narrowing of the gyrase N-gate coordinates T-segment capture and strand passage.

Project description:DNA gyrase introduces negative supercoils into DNA in an ATP-dependent reaction. DNA supercoiling is catalyzed by a strand-passage mechanism, in which a T-segment of DNA is passed through the gap in a transiently cleaved G-segment. Strand passage requires the coordinated closing and opening of three protein interfaces in gyrase, the N-gate, DNA-gate, and C-gate. We show here that DNA binding to the DNA-gate of gyrase and wrapping of DNA around the C-terminal domains of GyrA induces a narrowing of the N-gate. This half-closed state prepares capture of a T-segment in the upper cavity of gyrase. Subsequent N-gate closure upon binding of ATP then poises the reaction toward strand passage. The N-gate reopens after ATP hydrolysis, allowing for further catalytic cycles. DNA binding, cleavage, and wrapping and N-gate narrowing are intimately linked events that coordinate conformational changes at the DNA and the N-gate.

| S-EPMC3161603 | biostudies-literature

Friend or Foe: Protein Inhibitors of DNA Gyrase.

Project description:DNA gyrase is essential for the successful replication of circular chromosomes, such as those found in most bacterial species, by relieving topological stressors associated with unwinding the double-stranded genetic material. This critical central role makes gyrase a valued target for antibacterial approaches, as exemplified by the highly successful fluoroquinolone class of antibiotics. It is reasonable that the activity of gyrase could be intrinsically regulated within cells, thereby helping to coordinate DNA replication with doubling times. Numerous proteins have been identified to exert inhibitory effects on DNA gyrase, although at lower doses, it can appear readily reversible and therefore may have regulatory value. Some of these, such as the small protein toxins found in plasmid-borne addiction modules, can promote cell death by inducing damage to DNA, resulting in an analogous outcome as quinolone antibiotics. Others, however, appear to transiently impact gyrase in a readily reversible and non-damaging mechanism, such as the plasmid-derived Qnr family of DNA-mimetic proteins. The current review examines the origins and known activities of protein inhibitors of gyrase and highlights opportunities to further exert control over bacterial growth by targeting this validated antibacterial target with novel molecular mechanisms. Furthermore, we are gaining new insights into fundamental regulatory strategies of gyrase that may prove important for understanding diverse growth strategies among different bacteria.

| S-EPMC10886550 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data