Recognition of DNA Supercoil Geometry by<i>Mycobacterium tuberculosis</i>Gyrase

Ashley, Rachel E.; Blower, Tim R.; Osheroff, Neil

doi:10.1021/acs.biochem.7b00681

Cited by 17 publications

(32 citation statements)

References 58 publications

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“…Furthermore, the presence of this C-loop indirectly slightly modulates the DNAstimulated ATPase activity of Mtb DNA gyrase and has little impact on DNA-strand passage. This modulation could in part explain why the N-gate region is essential for the recognition of DNA geometry by Mtb gyrase, as was highlighted recently (Ashley et al, 2017). Therefore, our observations strengthen the idea that Mtb DNA gyrase must escape from the ''extremely open'' N-gate conformation before it can proceed onto the productive catalytic pathway.…”

Section: Discussionsupporting

confidence: 85%

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

et al. 2019

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Graphical Abstract Highlights d The N-gate of GyrBA57 (Mtb) gyrase lies in an ''extremely open'' conformation d The N-gate stays open in the crystal and in solution even in the presence of AMPPNP d The C-loop (a Corynebacteriales-specific GyrB insert) stabilized this conformation d This C-loop plays a role in the ATPase activity of the enzyme SUMMARYDespite sharing common features, previous studies have shown that gyrases from different species have been modified throughout evolution to modulate their properties. Here, we report two crystal structures of Mycobacterium tuberculosis DNA gyrase, an apo and AMPPNP-bound form at 2.6-Å and 3.3-Å resolution, respectively. These structures provide high-resolution structural data on the quaternary organization and interdomain connections of a gyrase (full-length GyrB-GyrA57) 2 thus providing crucial inputs on this essential drug target. Together with small-angle X-ray scattering studies, they revealed an ''extremely open'' N-gate state, which persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively the C-loop and DEEE-loop. We show that the C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Our results image a conformational state which might be targeted for drug discovery.

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Section: Discussionsupporting

confidence: 85%

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

et al. 2019

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“…This ~20-fold time difference between the two reactions likely reflects the acute temporal requirement to rapidly remove positive supercoils that accumulate in front of the replication fork as compared to the maintenance of steady state levels of negative DNA supercoiling. 63–64…”

Section: Resultsmentioning

confidence: 99%

Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase

Gibson¹,

et al. 2019

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Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 μM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 μM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or with intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound.

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“…44–47 A recent study by Ashley et al demonstrated that M. tuberculosis gyrase maintains 2-3-fold lower levels of cleavage complexes on positively compared to negatively supercoiled DNA in the absence or presence of fluoroquinolones. 48 This attribute makes gyrase a safer enzyme (for cells) to work ahead of DNA tracking systems, but may also affect the cytotoxicity of fluoroquinolones.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of Action of Mycobacterium tuberculosis Gyrase Inhibitors: A Novel Class of Gyrase Poisons

et al. 2018

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Tuberculosis is one of the leading causes of morbidity worldwide, and the incidences of drug resistance and intolerance are prevalent. Thus, there is a desperate need for the development of new antitubercular drugs. Mycobacterium tuberculosis gyrase inhibitors (MGIs) are napthyridone/aminopiperidine-based drugs that display activity against M. tuberculosis cells and tuberculosis in mouse models [Blanco, D., et al. (2015) Antimicrob. Agents Chemother. 59, 1868-1875]. Genetic and mutagenesis studies suggest that gyrase, which is the target for fluoroquinolone antibacterials, is also the target for MGIs. However, little is known regarding the interaction of these drugs with the bacterial type II enzyme. Therefore, we examined the effects of two MGIs, GSK000 and GSK325, on M. tuberculosis gyrase. MGIs greatly enhanced DNA cleavage mediated by the bacterial enzyme. In contrast to fluoroquinolones (which induce primarily double-stranded breaks), MGIs induced only single-stranded DNA breaks under a variety of conditions. MGIs work by stabilizing covalent gyrase-cleaved DNA complexes and appear to suppress the ability of the enzyme to induce double-stranded breaks. The drugs displayed little activity against type II topoisomerases from several other bacterial species, suggesting that these drugs display specificity for M. tuberculosis gyrase. Furthermore, MGIs maintained activity against M. tuberuclosis gyrase enzymes that contained the three most common fluoroquinolone resistance mutations seen in the clinic and displayed no activity against human topoisomerase IIα. These findings suggest that MGIs have potential as antitubercular drugs, especially in the case of fluoroquinolone-resistant disease.

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Recognition of DNA Supercoil Geometry byMycobacterium tuberculosisGyrase

Cited by 17 publications

References 58 publications

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase

Mechanism of Action of Mycobacterium tuberculosis Gyrase Inhibitors: A Novel Class of Gyrase Poisons

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