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

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

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Type II topoisomerase Type II topoisomerase challenges challenges Claudine MAYER Claudine MAYER Universit Paris Diderot Institut Pasteur Runion MASIM 2017 Presentation Presentation of the team f the team CNRS UMR 3528 Biologie

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Type II topoisomerase Type II topoisomerase challenges challenges

Claudine MAYER Claudine MAYER

Université Paris Diderot Institut Pasteur

Réunion MASIM 2017

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Presentation Presentation of the team f the team

LABORATORY / UNIT CNRS UMR 3528 DEPARTMENT Biologie structurale des processus cellulaires et maladies infectieuses CNRS UMR 3523 Unité de Chimie Organique

X‐ray crystallography Molecular biophysics and biochemistry Bacterial genetics Structural and mechanistic enzymology Mycobacterial proteins

Institut Pasteur, CNRS UMR 3528, University Paris Diderot

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Presentation Presentation of the team f the team

Head: Pedro M. Alzari (PR IP)

Structural studies of type II topoisomerases Crystallography, biophysics, cryoEM, modeling

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DNA topoisomerases DNA topoisomerases

topoisomerases

Manipulation and control of DNA topology

Helicase DNA polymerase Clamp loader Beta Clamp

In the cell, DNA is subjected to bending, folding, overwinding and underwinding

Replication Replication

relaxed supercoiled

Transcription Transcription Cell division Cell division

  • Unwinding to gain access
  • Removing supercoils
  • Separation of daughter chromosomes

DNA condensation DNA condensation

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DNA topoisomerases DNA topoisomerases

There are two types of topoisomerases Type I Type II Type II

Generic Generic type IIA type IIA topoisomerase topoisomerase 350-400 kDa 350-400 kDa 150-250 kDa 150-250 kDa

Modular Modular dynamic dynamic nanomachines nanomachines

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  • M. tuberculosis
  • M. tuberculosis DNA gyrase

DNA gyrase

Agra Agrawal et al et al, Bi , Bioche

  • chem J 20

2013 Pi Pito ton et al al, , Acta Acta cryst cryst F 20 2009 Pit Piton et

  • n et al,

al, PlosOne PlosOne 2010 010 Pit Piton et

  • n et al,

al, PlosOne PlosOne 2010 010 Boui Bouige ge et al et al, Bi , Bioch

  • chem J

J 201 2013 Da Darmo rmon et al al, , Acta Acta cryst cryst F 20 2012

3IFZ 3IFZ 3IG0 3IG0 3M4I 3M4I 4G3N 4G3N 3ZM7 3ZM7

Ro Roué et al et al, , Acta Acta cryst cryst F 20 2013

Structure-function Structure-function relationships relationships studies tudies of the 4 isolated

  • f the 4 isolated domains

domains

The unique type II topoisomerase in Mycobacterium tuberculosis Resistance Resistance to

  • fluoroquinolones

fluoroquinolones collaboration A. collaboration A. Aubry, UPMC Aubry, UPMC

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SLIDE 7
  • M. tuberculosis
  • M. tuberculosis DNA gyrase

DNA gyrase

Catalytic core

Jérémie PITON Thèse, 2010 Structural insights into quinolone resistance mechanism

ATPase domain

Mélanie ROUE ATER, 2011 Structural insights into ATP hydrolysis and its inhibition

CTD

Amélie DARMON Thèse, 2013 Structural insights into DNA wrapping mechanism

Structure‐function relationships of the 3 functional domains

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  • M. tuberculosis
  • M. tuberculosis DNA gyrase

DNA gyrase

The unique type II topoisomerase in Mycobacterium tuberculosis In silico In silico studies studies of FQR mechanisms

  • f FQR mechanisms

coll

  • llaboration G.

aboration G. André-Leroux, INRA André-Leroux, INRA

D9 D94 A90 A90 R4 R482

Wild type or mutants Wild type or mutants Atypical binding mode Atypical binding mode

Binding energies as a tool to predict resistance phenotype

fluoroquinolone

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Type IIB topoisomerases Type IIB topoisomerases

Topoisomerase VIII, a new member of the type IIB family Homology Homology modeling modeling collaboration P.

  • llaboration P. Forterre, UPSud

Forterre, UPSud & IP IP

Schematic multiple alignment

Plasmid maintenance and transfer

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Type IIB topoisomerases Type IIB topoisomerases

3D‐models built using homology modeling

2ZBK

pPpol Adeg Mmar

K450 hinge residue

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Type IIB topoisomerases Type IIB topoisomerases

Modeling of the heterotetramer

2Q2E

Many uncertainties concerning domain‐domain and subunit‐subunit interfaces!

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Type IIB topoisomerases Type IIB topoisomerases

The meiotic topoisomerase‐like complex Remote Remote homology homology modeling

  • deling

collaboration M. Grelon, INRA

  • llaboration M. Grelon, INRA

Double Strand Breaks Double Strand Breaks

The DSB are catalyzed by Spo11

Spo11 Spo11

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Type IIB topoisomerases Type IIB topoisomerases

The meiotic topoisomerase‐like complex In A. thaliana, 2 Spo11 proteins form a heterodimer that catalyse meiotic DSB

Does the Spo11 heterodimer need a partner?

A 493 aa protein highly conserved in flowering plant (Magnoliophyta)

GHKL GHKL Sm SmD Trans Transducer ucer Ct Cter er 183 183 1 266 266 432 432 493 493

MTOPVIB Highly conserved motifs Highly conserved motifs

HHPred searches detected structural homology Top6B

Genetic Genetic screen screen

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Type IIB topoisomerases Type IIB topoisomerases

The meiotic topoisomerase‐like complex

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Conclusion and perspectives Conclusion and perspectives

Experimental Experimental and and in silico in silico structural studies structural studies are highly are highly complementary

  • mplementary

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 Sequence‐structure‐pocket geometry relationships

Spo11 homodimerisation vs heterodimerisation Subunit A – subunit B interactions ATP-binding pocket and inhibition mechanisms

Sequence‐structure‐resistance phenotype relationships

Resistance phenotype = f(aa substitution, fluoroquinolone)

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Jérémie Piton Mélanie Roué Amélie Darmon Marcela França Penna Alexandre Guignard Thomas Zapf Cédric Pissis Geneviève Janvier Elodie Leroy Elisa Quiot Stéphanie Petrella Laboratoire de Bactériologie UPMC Alexandra Aubry and coworkers

UMS

Pedro Alzari

Institut Jean‐Pierre Bourgin, INRA Mathilde Grelon and coworkers

MaIAGE, INRA

Gwenaëlle André‐Leroux

Mti, Université Paris Diderot

Leslie Regad

BMGE, IP‐Université Paris Saclay Patrick Forterre and coworkers

PFBMI

Bertrand Raynal, Bruno Baron, Patrick England

PF6

Patrick Weber, Rafael Navaza, Ahmed Haouz

PTR n°367

Thank you

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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

  • Mayer. (2013). Mycobacterium tuberculosis DNA gyrase possesses two functional GyrA-
  • 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.