Type II topoisomerase Type II topoisomerase challenges challenges - PowerPoint PPT Presentation

Type II topoisomerase Type II topoisomerase challenges challenges Claudine MAYER Claudine MAYER Université Paris Diderot Institut Pasteur Réunion MASIM 2017

Presentation Presentation of the team f the team CNRS UMR 3528 Biologie structurale des processus cellulaires et maladies infectieuses DEPARTMENT CNRS UMR 3523 Unité de Chimie Organique Institut Pasteur, CNRS UMR 3528, University Paris Diderot LABORATORY / UNIT X ‐ ray crystallography Molecular biophysics and biochemistry Bacterial genetics Structural and mechanistic enzymology Mycobacterial proteins

Presentation Presentation of the team f the team Head : Pedro M. Alzari (PR IP) Structural studies of type II topoisomerases Crystallography, biophysics, cryoEM, modeling

DNA topoisomerases DNA topoisomerases In the cell, DNA is subjected to bending, folding, overwinding and underwinding Replication Replication Manipulation and Beta Clamp control of DNA topology DNA condensation DNA condensation Clamp loader Helicase DNA polymerase relaxed supercoiled topoisomerases Cell division Cell division  Unwinding to gain access Transcription Transcription  Removing supercoils  Separation of daughter chromosomes

DNA topoisomerases DNA topoisomerases There are two types of topoisomerases Modular dynamic Modular dynamic nanomachines nanomachines Type I Type II Type II 350-400 kDa 350-400 kDa 150-250 kDa 150-250 kDa Generic type IIA Generic type IIA topoisomerase topoisomerase

M. tuberculosis M. tuberculosis DNA gyrase DNA gyrase The unique type II topoisomerase in Mycobacterium tuberculosis Resistance to Resistance o fluoroquinolones fluoroquinolones collaboration A. Aubry, UPMC collaboration A. Aubry, UPMC Structure-function Structure-function relationships relationships studies tudies of the 4 isolated of the 4 isolated domains domains Ro Roué et al et al, , Acta Acta cryst cryst F 20 2013 Pito Pi ton et al al, , Acta Acta cryst cryst F 20 2009 Da Darmo rmon et al al, , Acta Acta cryst cryst F 20 2012 Piton et Pit on et al, al, PlosOne PlosOne 2010 010 Agra Agrawal et al et al, Bi , Bioche ochem J 20 2013 Pit Piton et on et al, al, PlosOne PlosOne 2010 010 Bouige Boui ge et al et al, Bi , Bioch ochem J J 201 2013 3ZM7 3ZM7 3IG0 3IG0 3M4I 3M4I 3IFZ 3IFZ 4G3N 4G3N

M. tuberculosis M. tuberculosis DNA gyrase DNA gyrase Structure ‐ function relationships of the 3 functional domains ATPase domain Catalytic core Mélanie ROUE Jérémie PITON ATER, 2011 Thèse, 2010 Structural insights into ATP Structural insights into quinolone hydrolysis and its inhibition resistance mechanism CTD Amélie DARMON Thèse, 2013 Structural insights into DNA wrapping mechanism

M. tuberculosis DNA gyrase M. tuberculosis DNA gyrase The unique type II topoisomerase in Mycobacterium tuberculosis In silico In silico studies studies of FQR mechanisms of FQR mechanisms coll ollaboration G. aboration G. André-Leroux, INRA André-Leroux, INRA Binding energies as a tool to predict resistance phenotype fluoroquinolone R4 R482 D94 D9 A90 A90 Wild type or mutants Wild type or mutants Atypical binding mode Atypical binding mode

Type IIB topoisomerases Type IIB topoisomerases Topoisomerase VIII, a new member of the type IIB family Homology Homology modeling modeling collaboration P. ollaboration P. Forterre, UPSud Forterre, UPSud & IP IP Plasmid maintenance and transfer Schematic multiple alignment

Type IIB topoisomerases Type IIB topoisomerases 3D ‐ models built using homology modeling 2ZBK Adeg Mmar pPpol K450 hinge residue

Type IIB topoisomerases Type IIB topoisomerases Modeling of the heterotetramer 2Q2E Many uncertainties concerning domain ‐ domain and subunit ‐ subunit interfaces!

Type IIB topoisomerases Type IIB topoisomerases The meiotic topoisomerase ‐ like complex Remote Remote homology homology modeling odeling collaboration M. Grelon, INRA ollaboration M. Grelon, INRA The DSB are catalyzed by Spo11 Double Double Spo11 Spo11 Strand Strand Breaks Breaks

Type IIB topoisomerases Type IIB topoisomerases The meiotic topoisomerase ‐ like complex Genetic screen Genetic screen A 493 aa protein highly conserved in In A. thaliana , 2 Spo11 proteins form a flowering plant ( Magnoliophyta ) heterodimer that catalyse meiotic DSB HHPred searches detected structural homology Top6B 1 183 183 266 266 432 432 493 493 GHKL GHKL Sm SmD Transducer Trans ucer Ct Cter er MTOPVIB Highly Highly conserved conserved motifs motifs Does the Spo11 heterodimer need a partner?

Type IIB topoisomerases Type IIB topoisomerases The meiotic topoisomerase ‐ like complex

Conclusion and perspectives Conclusion and perspectives Experimental Experimental and and in silico in silico structural studies structural studies are highly are highly complementary omplementary to to gain insight into gain insight into the structure of the structure of complex complex macromolecular acromolecular assemblies ssemblies Sequence ‐ structure ‐ solubility relationships Spo11 vs archaeal Top6A Sequence ‐ structure ‐ interaction relationships Spo11 homodimerisation vs heterodimerisation Subunit A – subunit B interactions Sequence ‐ structure ‐ pocket geometry relationships ATP-binding pocket and inhibition mechanisms Sequence ‐ structure ‐ resistance phenotype relationships Resistance phenotype = f(aa substitution, fluoroquinolone)

Thank you Jérémie Piton Mti, Université Paris Diderot UMS Mélanie Roué Leslie Regad Pedro Alzari Amélie Darmon Marcela França Penna Laboratoire de MaIAGE, INRA Alexandre Guignard Bactériologie Gwenaëlle André ‐ Leroux Thomas Zapf UPMC Cédric Pissis Alexandra Aubry BMGE, IP ‐ Université Paris Saclay Geneviève Janvier and coworkers Patrick Forterre and coworkers Elodie Leroy Elisa Quiot Stéphanie Petrella Institut Jean ‐ Pierre Bourgin, INRA Mathilde Grelon and coworkers PTR n°367 PFBMI Bertrand Raynal, Bruno Baron, Patrick England PF6 Patrick Weber, Rafael Navaza, Ahmed Haouz

Main publications Main publications A. Bouige, A. A. Darmon Darmon, J. J. Pi Piton ton, M. M. Rou Roué, S. S. Petrella Petrella, E. Capton, P. Forterre, A. Aubry, and C. C. Mayer. (2013). Mycobacterium tuberculosis DNA gyrase possesses two functional GyrA- Mayer boxes. Bioch Biochem J. , 455 455 (3) (3), 285-294. A. Agrawal, M. M. Rou Roué, C. Spitzfaden, S. S. Pe Petrell trella, A. Aubry, MM. Hann, B. Bax, and C. C. Mayer Mayer. (2013). Mycobacterium tuberculosis DNA gyrase ATPase domain structures suggest a dissociative mechanism that explains how ATP hydrolysis is coupled to domain motion. Bioch Biochem J. , 456 456 (2) (2), 263-273. C. C. Mayer Mayer and Y.L. Janin. (2014). Non-quinolone inhibitors of bacterial type IIA topoisomerases: a feat of bioisosterism. Chem Chem Rev., Rev., 114 114 (4) (4), 2313-2342. D. Gadelle, M. Krupovic, K. Raymann, C. C. Mayer Mayer, and P. Forterre. (2014). DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria. Nucle Nucleic Acids cids Res Res. , 42 (13) 13), 8578-8591. N. Vrielynck, A. Chambon, D. Vezon, L. Pereira, L. Chelysheva, A. De Muyt, C. Mézard, C. C. Mayer Mayer, and M. Grelon. (2016). A DNA topoisomerase VI-like complex initiates meiotic recombination. Scien Science , 351 351 (6276) (6276), 939-943.