Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

TY - JOUR

T1 - Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

AU - Felicori, Liza

AU - Jameson, Katie H.

AU - Roblin, Pierre

AU - Fogg, Mark J.

AU - Garcia-Garcia, Transito

AU - Ventroux, Magali

AU - Cherrier, Mickaël V.

AU - Bazin, Alexandre

AU - Noirot, Philippe

AU - Wilkinson, Anthony J.

AU - Molina, Franck

AU - Terradot, Laurent

AU - Noirot-Gros, Marie Françoise

PY - 2016/1/8

Y1 - 2016/1/8

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84959538035&partnerID=8YFLogxK

U2 - 10.1093/nar/gkv1318

DO - 10.1093/nar/gkv1318

M3 - Article

AN - SCOPUS:84959538035

VL - 44

SP - 450

EP - 463

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 1

ER -