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

Nucleic Acids Res. 2016 Jan 8;44(1):449-63. doi: 10.1093/nar/gkv1318. Epub 2015 Nov 28.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Bacillus subtilis / genetics
  • Bacillus subtilis / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • DNA Replication*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Intracellular Space
  • Models, Molecular
  • Molecular Sequence Data
  • Multiprotein Complexes / metabolism*
  • Mutation
  • Position-Specific Scoring Matrices
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping / methods
  • Protein Multimerization
  • Protein Transport
  • Sequence Alignment
  • Structure-Activity Relationship
  • Zinc / metabolism


  • Bacterial Proteins
  • DNA-Binding Proteins
  • Multiprotein Complexes
  • Zinc