Metal stoichiometry and functional studies of the diphtheria toxin repressor

24 March 2003

journal article
research article
Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences

Vol. 100 (7) , 3808-3813
https://doi.org/10.1073/pnas.0737977100

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 Fe²⁺ and not Fe³⁺ 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.

Keywords

This publication has 29 references indexed in Scilit:

Crystal Structure of a Cobalt-activated Diphtheria Toxin Repressor-DNA Complex Reveals a Metal-binding SH3-like Domain
Journal of Molecular Biology, 1999
Comparison of high‐resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation‐anion binding site
Protein Science, 1997
Identification of the primary metal ion-activation sites of the diphtheria tox represser by X-ray crystallography and site-directed mutational analysis
Nature Structural & Molecular Biology, 1996
High-Resolution Structure of the Diphtheria Toxin Repressor Complexed with Cobalt and Manganese Reveals an SH3-like Third Domain and Suggests a Possible Role of Phosphate as Co-corepressor^,
Biochemistry, 1996
Three-dimensional structure of the diphtheria toxin repressor in complex with divalent cation co-repressors
Structure, 1995
Analysis of diphtheria toxin repressor‐operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals
Molecular Microbiology, 1993
A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding
Analytical Biochemistry, 1976
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Analytical Biochemistry, 1976
Ferrozine---a new spectrophotometric reagent for iron
Analytical Chemistry, 1970