title: Metal stoichiometry and functional studies of the diphtheria toxin repressor.
abstract: 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.
authors.affiliation: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Spiering
authors.forename: Dagmar
authors.initials: D
authors.lastname: Ringe
authors.forename: John R
authors.initials: JR
authors.lastname: Murphy
authors.forename: Michael A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003353:Corynebacterium diphtheriae / Q000378:metabolism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D008670:Metals; D011994:Recombinant Proteins