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 -