dna gyrase is also known as

3a). conceived the study and designed the experiments; A.V.B. The ATPase domain is sitting 11 away from the DNA-binding/cleavage domain (Supplementary Fig. Med. 16, 2334 (2014). State 1 and 2 correspond to a conformational oscillation before the opening of the DNA-gate (Supplementary Movie2). The E. coli structure is reminiscent of a state immediately following G-segment binding and wrapping, a conformational intermediate that could have been trapped by gepotidacin. 37mg of GyrA were obtained from 3L of cultures. The overall RMSD between State 1 and State 2 conformations is of 2.2. In addition, the information derived from the thermophilic homolog does not account for all the mechanistic and structural specificities of the E. coli DNA gyrase, the genetic model for which functional data have been accumulated over the past decades. Interestingly, a recent single-molecule study identified a conformational state after G-segment binding, possibly similar to the E. coli complex and preceding the state in the catalytic cycle, a conformation favorable to the T-segment strand passage13. The highly conserved GyrA-box motif, QRRGGKG48, located on the first blade of the -pinwheels is essential for DNA wrapping (Fig. Type IIA topoisomerase inhibition by a new class of antibacterial agents. All together this suggests that the presence of the transducer interaction network in E. coli is important for ensuring allosteric coupling between ATP hydrolysis and DNA supercoiling activities. To obtain a final reconstruction of the full complex with well-defined densities for each domain, we performed 3D classification without alignment yielding several different classes. ISSN 2041-1723 (online). The full architecture of the complex in presence of a T-segment still needs to be determined to better understand the molecular determinants of DNA capture. Rudolph, M. G. & Klostermeier, D. Mapping the spectrum of conformational states of the DNA- and C-gates in Bacillus subtilis gyrase. Sci. elife 7, e41215 (2018). A first round of real-space refinement in PHENIX62 was performed using local real-space fitting and global gradient-driven minimization refinement. The mechanism by which gyrase is able to influence the topological state of . If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Biol. & Klostermeier, D. Gyrase containing a single C-terminal domain catalyzes negative supercoiling of DNA by decreasing the linking number in steps of two. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Wigley, D. B., Davies, G. J., Dodson, E. J., Maxwell, A. Article Commun. J. Mol. In contrast, the analysis of the E. coli structure shows that R286 is engaged in an interaction network with E264 and R316 anchoring together secondary structures of the transducer and providing rigidity to the transducer terminal helix (Fig. The gyrA, gyrB, grlA, and grlB genes were amplified from S. aureus ISP8 (also known as 8325-4 or RN450; kindly provided by J. J. Iandolo) genomic DNA by PCR . Laponogov, I. et al. The density of the flexible loops connecting the ATPase domain to the TOPRIM domain are well defined allowing to position these elements crossing on top of the G-segment in the DNA-bound gyrase conformation (Fig. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity. Heymann, J. Ward, D. & Newton, A. & Mondragon, A. CryoEM structures of open dimers of gyrase A in complex with DNA illuminate mechanism of strand passage. Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Protein Sci. Department of Integrated Structural Biology, Institut de Gntique et de Biologie Molculaire et Cellulaire (IGBMC), 1 Rue Laurent Fries, 67404, Illkirch Cedex, France, Arnaud Vanden Broeck,Christophe Lotz,Julio Ortiz&Valrie Lamour, Centre National de Recherche Scientifique (CNRS) UMR 7104, Illkirch, France, Institut National de Sant et de Recherche Mdicale (INSERM) U1258, Illkirch, France, Universit de Strasbourg, Illkirch, France, Hpitaux Universitaires de Strasbourg, 1 Place de lHpital, 67091, Strasbourg Cedex, France, You can also search for this author in The complete DNA binding around the -pinwheel surface generates a twisting of ~200 consistent with the reported wrapping around DNA gyrase pinwheel blades47. 10d). 4c, d). Tth K284 at the same position points toward the N-gate central cavity and is not engaged in an hydrogen bond network since E264 is replaced by A262 and R316 by a L31438 (Fig. Free Full-Text | DNA Gyrase as a Target for Quinolones The first contact between DNA and the -pinwheel occurs at blade 3, wraps around the disk by contacting blade 4, 5, 6 and exits the -pinwheel through contact with blade 1. b Different views of the 6.3 cryo-EM map (in grey) zoomed on the -pinwheel wrapped with DNA and the corresponding molecular models in surface representation (same color code as in Fig. Natl Acad. These molecules and in particular gepotidacin, represent a promising alternative and are currently in clinical trial for the treatment of bacterial infections23,24,25. This was also observed in the T. thermophilus gyrase complex with DNA, where 130 bp out of 155 bp could be docked in the cryo-EM map albeit of a lower resolution 12. CAS Chem. J. Biol. Bioorg. Further information on research design is available in theNature Research Reporting Summary linked to this article. 304, 3136 (2014). Acta Crystallogr. 1 and Supplementary Fig. Crystallogr. 1b). 152, 3651 (2005). CAS Overexpression was performed in E. coli BL21 (DE3) pRARE2 in LB medium containing 50g/mL kanamycin and 35g/mL chloramphenicol. Overexpression was performed in E. coli BL21 (DE3) pRARE2 in LB medium containing 50g/mL kanamycin and 35g/mL chloramphenicol. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. Biol. Negative and positive controls are shown as relaxed (Rlx) or negatively supercoiled DNA species (SC), respectively. DNA topoisomerases are a class of enzymes that can change the topological state of closed-circular DNA molecules. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The drugs are selective for bacteria because the enzyme in bacteria is structurally different from that in mammals. Consequently, the first contact between the G-segment and the -pinwheel occurs at blade 3, then wraps around the -pinwheel by contacting blade 4, 5, 6 and exits the -pinwheel through contact with blade 1 containing the GyrA-box motif (Fig. The continuous density of the ATPase and DNA-binding/cleavage domains EM map at 5.9 allowed us to build unambiguously the protein linkers between the C-terminal end of the transducer helices of the ATPase domain and the N-terminal end of the TOPRIM domain (Fig. Nature 466, 935940 (2010). Nat. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. The subsequent 11-binned particles stack was refined in RELION2 without mask until late refinement iterations where a soft mask was applied to improve resolution. Cell 77, 609616 (1994). After discarding the poor-quality models, the remaining ab-initio model resulted in a final dataset of 191,456 particles, with a class probability threshold of 0.9 (Supplementary Fig. State 1 was solved at 4.0 using 60,548 particles and State 2 was solved at 4.6 using 53,655 particles (Supplementary Fig. Vanden Broeck, A., Lotz, C., Ortiz, J. et al. contracts here. Google Scholar. Sci. We could also build the linker between the C-terminal end of the GyrA coiled-coil domains and the -pinwheels on each side (Fig. CAS . Google Scholar. The 36bp DNA duplex and gepotidacin are shown in the bottom panel. The -pinwheel blades are numbered from 1 to 6. Tretter, E. M. & Berger, J. M. Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. ADS Antimicrob. The high-resolution features of the DNA-binding/cleavage domain structure now makes it possible to detect the density of small molecules and possibly catalytic ions in the cryo-EM map. 66, 213221 (2010). Proc. Model coordinates and density maps are available in the Protein Data Bank (PDB ID 6RKS [https://www.rcsb.org/structure/6RKS], 6RKU [https://www.rcsb.org/structure/6RKU], 6RKV [https://www.rcsb.org/structure/6RKV], 6RKW [https://www.rcsb.org/structure/6RKW]) and the EM Data Bank (EMD-4909 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4909], EMD-4910 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4910], EMD-4912 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4912], EMD-4913 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4913], EMD-4914 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4914], EMD-4915 [https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4915]). The GHKL region of the ATPase domain shows a strong sequence identity across species due to the universal conservation of the catalytic motifs, with more variability across the transducer sequence (Supplementary Fig. Despite the high flexibility of the complex, we were able to solve a structure of the gyrase DNA-binding/cleavage domain in the closed complex with DNA and gepotidacin at 4.0 resolution using a focused refinement strategy (Supplementary Fig. The overall DNA-bound DNA gyrase complex was solved at 6.6 using 94,633 particles. One class of 45,040 particles with a well-defined density of the GyrA -pinwheel was selected, followed by re-extraction of 11-binned particles yielding particles with pixel size of 0.88/pixel. Get the most important science stories of the day, free in your inbox. Stanger, F. V., Dehio, C. & Schirmer, T. Structure of the N-terminal Gyrase B fragment in complex with ADPPi reveals rigid-body motion induced by ATP hydrolysis. To obtain 2b). Reversal of this scheme relaxes DNA, and this mechanism also accounts for the ability of gyrase to catenate and uncatenate DNA rings. Atomic model and constraint dictionary of gepotidacin (GSK-2140944) were generated with the Grade server (http://grade.globalphasing.org). DNA gyrase, also known as DNA topoisomerase II, plays a critical role in bacterial cell survival, and hence has been used as a successful drug target for antibacterial chemotherapy . Several biochemical studies have shown that the CTD tail stimulates DNA wrapping coupled with ATP hydrolysis43,52. J. Biotechnol. Figure 1.6.5: Type II Isomerase activity. bacterial cell su rvival, and hence has been . Chem. The 130bp DNA duplex is chirally wrapped around the -pinwheel orienting the T-segment in the DNA-gate groove formed by the TOPRIM and tower domains to form a 60 angle positive crossover when T-segment path is extrapolated. Herein, we report the complete structure of the E. coli DNA gyrase nucleoprotein complex trapped by the antibiotic gepotidacin, using phase-plate single-particle cryo-electron microscopy. Ultramicroscopy 135, 2435 (2013). Gepotidacin (GSK2140944, purchased at MedChemExpress) resuspended at 10mM in 100% DMSO was added to reach a final concentration of 170M (1.7% DMSO). Elution was performed with the lysis buffer containing 250mM imidazol pH 8.0 and eluted proteins were directly attached on a 10ml Streptavidin Sepharose (GE Healthcare). CAS Peer reviewer reports are available. Finally, 8mM CHAPSO (SigmaAldrich) was added to the complex. We review known gyrase-specific drugs and toxins and . 475, 373398 (2018). Methods 9, 853854 (2012). 2007).The details of this mechanism are still under investigation, but a model, generically known as the "two-gate mechanism" (Roca and Wang 1992, 1994), is strongly supported by biochemical and . The superimposition shows the upward movement of the TOPRIM domain while the tower domain remains fixed. Biol. & Moore, P. B. It is not clear at this stage if DNA capture and transport is directly linked to the basic residues in the transducer and at which catalytic step molecular interaction with the T-segment intervenes.
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