Breaking and Restoring the Hydrophobic Core of a Centromere-binding Protein

Sadia Saeed, Thomas A Jowitt, Jim Warwicker, Finbarr Hayes

Research output: Contribution to journalArticlepeer-review


The ribbon-helix-helix (RHH) superfamily of DNA-binding proteins is dispersed widely in procaryotes. The dimeric RHH fold is generated by interlocking of two monomers into a 2-fold symmetrical structure that comprises four α-helices enwrapping a pair of antiparallel β-strands (ribbon). Residues in the ribbon region are the principal determinants of DNA binding, whereas the RHH hydrophobic core is assembled from amino acids in both the α-helices and ribbon element. The ParG protein encoded by multiresistance plasmid TP228 is a RHH protein that functions dually as a centromere binding factor during segrosome assembly and as a transcriptional repressor. Here we identify residues in the α-helices of ParG that are critical for DNA segregation and in organization of the protein hydrophobic core. A key hydrophobic aromatic amino acid at one position was functionally substitutable by other aromatic residues, but not by non-aromatic hydrophobic amino acids. Nevertheless, intramolecular suppression of the latter by complementary change of a residue that approaches nearby from the partner monomer fully restored activity in vivo and in vitro. The interactions involved in assembling the ParG core may be highly malleable and suggest that RHH proteins are tractable platforms for the rational design of diverse DNA binding factors useful for synthetic biology and other purposes.
Original languageEnglish
Pages (from-to)9273-9283
Number of pages10
JournalThe Journal of biological chemistry
Issue number14
Publication statusPublished - 2015


  • Escherichia coli (E. coli)
  • antibiotic resistance
  • centromere-binding protein
  • microbiology
  • plasmid
  • protein folding
  • ribbon-helix-helix


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