dna gyrase is also known as

Images of the DNA gyrase complex were recorded using a Titan Krios with a Volta Phase Plate at a 500nm defocus target, allowing a significant increase in contrast of the images and optimization of the image acquisition rate27,28 (Fig. 196 Another related enzyme, . A half-map cross-validation was performed to define 1.5 as the best refinement weight in PHENIX allowing atom clash reduction and prevention of model overfitting (Supplementary 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 . Biology Genetics Genetics chp. J. Biol. c Selection of 2D classes from reference-free 2D classification. Basu, A., Parente, A. C. & Bryant, Z. Bioorg. Chen, S. F. et al. DNA sequence was modified according to the DNA used in our structure. Danev, R. & Baumeister, W. Cryo-EM single particle analysis with the Volta phase plate. 475, 373398 (2018). Nat. Mycobacterium tuberculosis DNA gyrase as a target for drug - PubMed Gyrase is a tetramer of the form A 2 B 2.The tetramer has two chambers that can accommodate two strands of DNA. Biol. ADS ATP hydrolysis is measured by following the oxidation of NADH mediated by pyruvate kinase (PK) and lactate dehydrogenase (LDH). 3c). bacterial dna gyrase: Topics by Science.gov Schmidt, B. H., Osheroff, N. & Berger, J. M. Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity. 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. Appl. J. Struct. 6a, b and Supplementary Fig. 5, 570581 (2019). The spatial arrangement of the -pinwheels induces an overall ~150 bending of the G-segment in the E. coli gyrase. Briefly, the nucleic acids were dissolved in DNAse-free water at 1mM concentration. Lett. Elife 5, e13046 (2016). DNA gyrase and topoisomerase IV are the biological targets of the quinolones in bacterial cells. PubMed J. Biol. 12, 827841 (2011). Commun. Finally, B-factors were refined by a final round of real-space refinement in PHENIX using the same settings as before. J. Struct. 4a). 275, 94689475 (2000). Atomic model and constraint dictionary of gepotidacin (GSK-2140944) were generated with the Grade server (http://grade.globalphasing.org). As gyrase is essential in prokaryotes, it is a good target for antibacterial agents. However, the use of any antibiotics may lead to the appearance of mutations and the development of resistant strains26. 3). Provided by the Springer Nature SharedIt content-sharing initiative. 4b). The highly conserved GyrA-box motif, QRRGGKG48, located on the first blade of the -pinwheels is essential for DNA wrapping (Fig. Nucleic Acids Res. 35). https://doi.org/10.1038/s41467-019-12914-y. The 2 conformations mostly differ in the GyrB subunit, more particularly the TOPRIM insertion that rotates around the GyrA N-terminal arm with a 5.0 upward displacement (Fig. 2). PubMed Then, 20 nucleic acids from the structure of S. aureus DNA gyrase41 (PDB 5IWM) were copied and fitted into our atomic model. 1). Biophys. DNA Gyrase - an overview | ScienceDirect Topics Commun. Other structural elements missing in crystal structures such as surface loops, -helices of the DNA binding/cleavage and C-gate domains could be completed or corrected in the GyrB and GyrA subunits representing more than 5% of the total sequence (Supplementary Fig. After 30min, reactions were stopped by addition of SDS 1%. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. This leads to the positioning of the GyrA-box (in magenta) at the exit of the DNA path around the -pinwheel, in close contact with DNA. USA 93, 40574062 (1996). The ab-initio model was low-pass filtered to 30 and was used as a reference for homogeneous refinement in cryoSPARC resulting in a 5.4 map. Then, a copy of the first -pinwheel was fitted into the density of the second -pinwheel in COOT. 13b). DNA gyrase is a remarkable enzyme, catalysing the seemingly complex reaction of DNA supercoiling. b Gepotidacin binding site. Biotechnol. 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. High-resolution features of the DNA-binding/cleavage domain bound to gepotidacin. The phylogenetic tree was generated by neighbour-joining method using the multiple alignment in Clustal Omega. 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. 25, 16051612 (2004). Our data unveil the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. Mastronarde, D. N. & Held, S. R. Automated tilt series alignment and tomographic reconstruction in IMOD. The modified pET28b used for wild-type T. thermophilus GyrB overexpression12 was mutated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent) in order to generate two plasmids harboring K284R or K284Q mutations (Supplementary Table4). Consequently, the high degree of structural conservation of the bacterial and the human Top2 suggests that this mechanism of sliding and swiveling can be extended to the bacterial Topo IIA, and thus to DNA gyrase. The K284 residue is not engaged in an interaction network. UCSF Chimeraa visualization system for exploratory research and analysis. 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 Nat. volume10, Articlenumber:4935 (2019) Structural insights into the gating of DNA passage by the topoisomerase II DNA-gate. In T. thermophilus, the equivalent position K284 is pointing in the cavity and could potentially mediate an interaction with DNA; however, our experiments show that point mutations have no effect on the catalytic activities. elife 7, e41215 (2018). Oxolinic acid is known to stabilize an enzymeDNA complex, in which DNA remains cleaved (Gellert et al., 1978; . To allow better positioning and refinement of the atomic models in the EM density of the overall structure, we also solved three additional structures with better defined densities for the ATPase domain and DNA-binding/cleavage domain (5.9), the DNA-binding/cleavage domain without the TOPRIM insertion (4.0) and the DNA-binding/cleavage domain with the C-terminal -pinwheels wrapped by DNA (6.3) (Supplementary Figs. & Wang, J. C. DNA transport by a type II DNA topoisomerase: evidence in favor of a two-gate mechanism. Structural dynamics and mechanochemical coupling in DNA Gyrase. 46, 67736784 (2018). Methods 9, 853854 (2012). & Belnap, D. M. Bsoft: image processing and molecular modeling for electron microscopy. The absorbance was monitored at 340nm over 600s at 37C with a Shimadzu 1700 spectrophotometer. J. Struct. Finally, a focused 3D auto-refinement was performed in RELION2 using a soft mask around the DNA-binding/cleavage domain. 14). A dimeric atomic model of the DNA-binding/cleavage domain was generated using PDB 3NUH in PyMol (Schrodinger L.L.C.). Delgado, J. L., Hsieh, C. M., Chan, N. L. & Hiasa, H. Topoisomerases as anticancer targets. Finally, the gepotidacin molecule was added to the complex, which was further stabilized using ADPNP, a non-hydrolysable analog of ATP. An ab-initio 3D model was calculated with a final stack of 191,456 particles using cryoSPARC31 (Supplementary Fig. DNA replication is semiconservative, meaning that each strand in the DNA double helix acts as a template for the synthesis of a new, complementary strand. Nucleic Acids Res. Mol. The subsequent atomic model containing the ATPase domain, the DNA-binding/cleavage domain and one -pinwheel was rigid-body fitted into the overall complex structure solved at 6.6 using Chimera. 4d and Supplementary Fig. Thermal denaturation experiments on the E. coli mutant proteins using differential scanning fluorimetry showed the same behavior as the WT protein except for the E264A that shows a decreased thermal stability (Supplementary Fig. Antimicrob. The resulting atomic model was stripped of all ions and water molecules, with all occupancies set to 1 and B-factors set to 50. Chem. Ward, D. & Newton, A. & Berger, J. M. The structure of DNA-bound human topoisomerase II alpha: conformational mechanisms for coordinating inter-subunit interactions with DNA cleavage. https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4909, https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4910, https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4912, https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4913, https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4914, https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4915, https://doi.org/10.1128/ecosalplus.ESP-0010-2014, Description of Additional Supplementary Files, http://creativecommons.org/licenses/by/4.0/, Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin, Structural basis for allosteric regulation of Human Topoisomerase II, Reliable identification of protein-protein interactions by crosslinking mass spectrometry, Cancel J. Conformational changes of the DNA-gate from closed to pre-opening state may conduct a signal to the -pinwheel through the tethered acidic tail promoting a -pinwheel vertical movement. J. Comput Chem. a Superimposition of the cleavage complex in a closed state (this study) to the DNA-free DNA-binding/cleavage domain (apo state, PDB ID 3NUH). Sci. The spatial arrangement of the . 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. 425, 26322640 (2013). Computational resources were provided by the Mso-centre de Calcul (University of Strasbourg). CAS Brino, L. et al. Overexpression and Purification of Bacterial DNA Gyrase PHENIX: a comprehensive Python-based system for macromolecular structure solution. 10). Cell 77, 609616 (1994). Crystallogr. The sample was centrifuged 2h at 16,000g to remove potential aggregates. Nature 351, 624629 (1991). Besides, MD simulation of the T-segment crossing through the open DNA-gate suggested the presence of a conformation where the central cavity is widened and flattened, a state previously observed in a crystal structure of the B. subtilis DNA gyrase45. and V.L. 12). Chem. Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase. A model is shown in Figure 1 (left side). Corbett, K. D., Shultzaberger, R. K. & Berger, J. M. The C-terminal domain of DNA gyrase A adopts a DNA-bending beta-pinwheel fold. 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. 304, 3136 (2014). Nature Communications (Nat Commun) 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 . Both 10-His tag and Twin-strep tag were subsequently cleaved by PreScission protease (P3C) and Tobacco Etch Virus (TEV) cleavage (mass ratio 1:1:50 P3C-TEV-GyrA) overnight at 4C. Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase. 7 and Supplementary Table3). Natl Acad. Crystal structures of DNA-binding/cleavage domains in different states have led to propose that the DNA-gate opening is achieved by a sliding and swiveling motion of the two halves against each other, breaking the G-segment axis2,3,39,44. The usefulness of gyrase as a target of antibacterial agents stems from its mechanism of supercoiling (Schoeffler and Berger 2008; Nollmann et al. Sci. 10, 845858 (2015). These molecules and in particular gepotidacin, represent a promising alternative and are currently in clinical trial for the treatment of bacterial infections23,24,25. antibacterial chemotherapy (1 . The source data underlying Figs. A last round of 2D classification was then performed yielding a final particle stack of 338,616 particles. Four times binned particles from each dataset were separately subjected to two rounds of 2D classification in RELION2 to remove junk particles and contaminations resulting in a total of 479,667 particles for further processing. Google Scholar. Altogether this work paves the way for the structure determination of gyrase complexes with additional antibiotics using cryo-EM and the in-depth analysis of its allosteric regulation. Wendorff, T. J., Schmidt, B. H., Heslop, P., Austin, C. A. 66, 486501 (2010). Dynamic coupling between conformations and nucleotide states in DNA gyrase. Basu, A. et al. Collin, F., Karkare, S. & Maxwell, A. Exploiting bacterial DNA gyrase as a drug target: current state and perspectives. The type IIA DNA topoisomerases (Top2) are nanomachines that control DNA topology during multiple cellular processes such as replication, transcription and cell division1,2,3,4. GyrA was then further purified by an anion exchange chromatography step using a HiTrap Q HP column (GE Healthcare). 6b). The ATPase and DNA supercoiling activities of the Tth K284R and K284Q mutants remain in the same range as the WT at 37C (Fig. Electron density of the DNA and gepotidacin are shown in blue mesh. Rudolph, M. G. & Klostermeier, D. Mapping the spectrum of conformational states of the DNA- and C-gates in Bacillus subtilis gyrase. Gellert, M., Mizuuchi, K., ODea, M. H. & Nash, H. A. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Biol. Gepotidacin was then manually fitted in the empty electron density in COOT and then duplicated. Crystallogr. 130bp of the double-stranded DNA out of 180bp could be fitted and the corresponding base pairs were submitted in refinement in the cryo-EM map. The combination of the high bending angle of the G-segment (~150) with the peculiar orientation of the -pinwheels positions the T-segment to access the DNA-gate with an almost null or negative angle of ~10 (Supplementary Fig. 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. 5 and Supplementary Movie1). 287, 1863618644 (2012). Zhang, K. Gctf: Real-time CTF determination and correction. Exploiting bacterial DNA gyrase as a drug target: current DNA Wrapping around the GyrA CTD -pinwheel and GyrA-box structure. elife 5,e18722 (2016). The mutant proteins were recombinantly produced with no detectable denaturation. The GyrA-box motifs of each -pinwheel are located at the exit of the DNA path around the -pinwheel and act as clamps stabilizing the DNA curvature. Cell Biol. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Both duplicates were set to 0.5 occupancy. PubMed The ATPase-Core structure solved at 5.9 was used to rigid-body fit in Chimera the ATPase domain crystal structure in complex with ADPNP32 (PDB 1EI1) and the DNA-binding/cleavage domain refined in closed conformation. This is necessary because the unzipping of the DNA by helicase also unwinds it (since it is a double helix) and causes the introduction of positive supercoiling. The 10 missing amino acids (564574) following the GyrA-box were added using the modelling server Phyre264. 6a). 3). A double-nicked 180bp DNA duplex was reconstituted using 2 phosphorylated asymmetric synthetic oligonucleotides12 obtained from SigmaAldrich (Supplemental Table1). To obtain Article 1: There were three models suggested for DNA replication. This explains the role of the GyrA-box behaving as a at the end of the wrap mechanism, consistent with previous studies50,51. This motion increases the distance between GyrA Tower and GyrB TOPRIM from 8 to 11, inducing a slight shortening of the distance between the catalytic tyrosines from 26.4 to 25.6 and inducing the stretching of the G-segment by 2.5 in both directions (Fig. Refinement parameters, model statistics and validation scores are summarized in Supplementary Table2. and JavaScript. Is DNA gyrase found in humans? Chan, P. F. et al. D. Biol. You are using a browser version with limited support for CSS. Article Only one monomer of GyrB and GyrA is displayed for clarity. The mechanism by which gyrase is able to influence the. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. 66, 213221 (2010). Sample preparation and image acquisition. Proc. 26, 122129 (2017). As a consequence, this configuration seems unfavorable to T-segment strand passage and would require a structural rearrangement of the -pinwheel to proceed further. Rev. The atomic model of the overall structure has been deposited in the Protein Data Bank under accession numbers 6RKW. USA 73, 38723876 (1976). 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. 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. 4a). PubMed Free Full-Text | DNA Gyrase as a Target for Quinolones Biol. DNA Gyrase - notes - DNA Gyrase Prokaryotic topoisomerase II, also J. Med. 6c). Deletion of the insertion was shown to greatly reduce the DNA binding, supercoiling and DNA-stimulated ATPase activities of E. coli DNA gyrase17. Nat. Other data are available from the corresponding author upon reasonable request. Such flexibility of the DNA-binding/cleavage domain in presence of gepotidacin has also been observed in the S. aureus DNA crystal structures of DNA-binding/cleavage domains with an intact or doubly nicked DNA25. The overall DNA-bound DNA gyrase complex was solved at 6.6 using 94,633 particles.
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