Genetic and biophysical studies of diphtheria toxin repressor (DtxR) and the hyperactive mutant DtxR(E175K) support a multistep model of activation.
The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is the prototypic member of a superfamily of transition metal ion-activated transcriptional regulators that have been isolated from Gram-positive prokaryotes. Upon binding divalent transition metal ions, the N-terminal domain of DtxR undergoes a dynamic structural organization leading to homodimerization and target DNA binding. We have used site-directed mutagenesis and NMR analysis to probe the mechanism by which apo-DtxR transits from an inactive to a fully active repressor upon metal ion binding. We demonstrate that the ancillary metal-binding site mutant DtxR(H79A) requires higher concentrations of metal ions for activation both in vivo and in vitro, providing a functional correlation to the proposed cooperativity between ancillary and primary binding sites. We also demonstrate that the C-terminal src homology 3 (SH3)-like domain of DtxR functions to modulate repressor activity by (i) binding to the polyprolyl tether region between the N- and C-terminal domains, and (ii) destabilizing the ancillary binding site, leading to full inactivation of the repressor. Finally, we show by NMR analysis that the hyperactive phenotype of DtxR(E175K) results from the stabilization of a structural intermediate in the activation process. Taken together, the data presented support a multistep model for the activation of apo-DtxR by transition metal ions.
Department of Microbiology, Boston University School of Medicine, 650 Albany Street, X830, Boston, MA 02118, USA.
John F
JF
Love
Johanna C
JC
vanderSpek
Vedrana
V
Marin
Luis
L
Guerrero
Timothy M
TM
Logan
John R
JR
Murphy
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003353:Corynebacterium diphtheriae / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D000409:Alanine