title: Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners.
abstract: YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.
authors.affiliation: Departamento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, UFMG, 31270-901, Belo Horizonte, MG, Brazil Sys2Diag FRE3690-CNRS/ALCEDIAG, Montpellier, France.
authors.forename: Liza
authors.initials: L
authors.lastname: Felicori
authors.affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK.
authors.forename: Katie H
authors.initials: KH
authors.lastname: Jameson
authors.affiliation: Synchrotron SOLEIL-L'Orme des Merisiers Saint-Aubin- BP 48 91192 GIF-sur-YVETTE CEDEX, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Roblin
authors.affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Fogg
authors.affiliation: INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France AgroParisTech, UMR1319 Micalis, F-78350 Jouy-en-Josas, France.
authors.forename: Transito
authors.initials: T
authors.lastname: Garcia-Garcia
authors.affiliation: INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France AgroParisTech, UMR1319 Micalis, F-78350 Jouy-en-Josas, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Ventroux
authors.affiliation: CNRS, UMR 5086 Bases Moléculaires et Structurales de Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367 Lyon, France Université de Lyon, F-69622 Lyon, France Université Claude Bernard Lyon 1, F-69622 Villeurbanne, France.
authors.forename: Mickaël V
authors.initials: MV
authors.lastname: Cherrier
authors.affiliation: CNRS, UMR 5086 Bases Moléculaires et Structurales de Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367 Lyon, France Université de Lyon, F-69622 Lyon, France Université Claude Bernard Lyon 1, F-69622 Villeurbanne, France.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Bazin
authors.affiliation: INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France AgroParisTech, UMR1319 Micalis, F-78350 Jouy-en-Josas, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Noirot
authors.affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Wilkinson
authors.affiliation: Sys2Diag FRE3690-CNRS/ALCEDIAG, Montpellier, France franck.molina@sysdiag.cnrs.fr.
authors.forename: Franck
authors.initials: F
authors.lastname: Molina
authors.affiliation: CNRS, UMR 5086 Bases Moléculaires et Structurales de Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, 7 Passage du Vercors, F-69367 Lyon, France Université de Lyon, F-69622 Lyon, France Université Claude Bernard Lyon 1, F-69622 Villeurbanne, France laurent.terradot@ibcp.fr.
authors.forename: Laurent
authors.initials: L
authors.lastname: Terradot
authors.affiliation: INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France AgroParisTech, UMR1319 Micalis, F-78350 Jouy-en-Josas, France marie-francoise.gros@jouy.inra.fr.
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Noirot-Gros
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042541:Intracellular Space; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D009154:Mutation; D056510:Position-Specific Scoring Matrices; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods; D055503:Protein Multimerization; D021381:Protein Transport; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D015032:Zinc