Peptide inhibitors identify roles for SSB C-terminal residues in SSB/exonuclease I complex formation

Biochemistry. 2009 Jul 28;48(29):6764-71. doi: 10.1021/bi900361r.

Abstract

Bacterial single-stranded (ss) DNA-binding proteins (SSBs) facilitate DNA replication, recombination, and repair processes in part by recruiting diverse genome maintenance enzymes to ssDNA. This function utilizes the C-terminus of SSB (SSB-Ct) as a common binding site for SSB's protein partners. The SSB-Ct is a highly conserved, amphipathic sequence comprising acidic and hydrophobic elements. A crystal structure of Escherichia coli exonuclease I (ExoI) bound to a peptide comprising the E. coli SSB-Ct sequence shows that the C-terminal-most SSB-Ct Phe anchors the peptide to a binding pocket on ExoI and implicates electrostatic binding roles for the acidic SSB-Ct residues. Here, we use SSB-Ct peptide variants in competition experiments to examine the roles of individual SSB-Ct residues in binding ExoI in solution. Altering the C-terminal-most Pro or Phe residues in the SSB-Ct strongly impairs SSB-Ct binding to ExoI, confirming a major role for the hydrophobic SSB-Ct residues in binding ExoI. Alteration of N-terminal SSB-Ct residues leads to changes that reflect cumulative electrostatic binding roles for the Asp residues in SSB-Ct. The SSB-Ct peptides also abrogate SSB stimulation of ExoI activity through a competitive inhibition mechanism, indicating that the peptides can disrupt ExoI/SSB/ssDNA ternary complexes. Differences in the potency of the SSB-Ct peptide variants in the binding and nuclease inhibition studies indicate that the acidic SSB-Ct residues play a more prominent role in the context of the ternary complex than in the minimal ExoI/SSB-Ct interaction. Together, these data identify roles for residues in the SSB-Ct that are important for SSB complex formation with its protein partners.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism*
  • Exodeoxyribonucleases / antagonists & inhibitors
  • Exodeoxyribonucleases / chemistry
  • Exodeoxyribonucleases / metabolism*
  • Models, Molecular
  • Peptides / pharmacology*
  • Protein Binding
  • Protein Conformation

Substances

  • DNA-Binding Proteins
  • Enzyme Inhibitors
  • Escherichia coli Proteins
  • Peptides
  • SSB protein, E coli
  • Exodeoxyribonucleases
  • exodeoxyribonuclease I