Metal stoichiometry and functional studies of the diphtheria toxin repressor

Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3808-13. doi: 10.1073/pnas.0737977100. Epub 2003 Mar 24.


Diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor in Corynebacterium diphtheriae. DtxR is an iron sensor; metal-bound DtxR represses transcription of genes downstream of the tox operator. Wild-type DtxR [DtxR(wt)] and several mutant forms were overexpressed and purified from Escherichia coli. DtxR was isolated without bound metal. Metal reconstitution gave a binding stoichiometry of 2 per monomer for DtxR(wt) and 1 per monomer for DtxR(H79A) and DtxR(M10A). DNA binding of DtxR(H79A) and DtxR(M10A) indicates that metal site 2 is essential for activity. Metal binding lowers the dimerization K(d) of DtxR from low micromolar to 33 nM. Gel electrophoretic mobility-shift assays show that Fe(2+) and not Fe(3+) activates DtxR for DNA binding. This finding suggests that gene regulation by DtxR may be sensitive not only to iron levels but also to redox state of the iron. Mutations in the tox operator sequence indicate that DtxR dimers binding to DNA may be highly cooperative.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Binding Sites
  • Circular Dichroism
  • Corynebacterium diphtheriae / metabolism
  • DNA Primers
  • DNA, Bacterial / chemistry*
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Genetic Vectors
  • Metals / metabolism*
  • Models, Molecular
  • Molecular Weight
  • Nucleic Acid Conformation
  • Polymerase Chain Reaction
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Surface Plasmon Resonance


  • Bacterial Proteins
  • DNA Primers
  • DNA, Bacterial
  • DNA-Binding Proteins
  • DtxR protein, Corynebacterium diphtheriae
  • Metals
  • Recombinant Proteins