Mechanism of metal ion activation of the diphtheria toxin repressor DtxR.
The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10(-7), binding site 2 (primary) is a low-affinity binding site with a binding constant of 6.3 x 10(-4). These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.
D'Aquino
J Alejandro
JA
Department of Chemistry, Brandeis University, Waltham, MA 02454, USA.
Tetenbaum-Novatt
Jaclyn
J
White
Andre
A
Berkovitch
Fred
F
Ringe
Dagmar
D
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002151:Calorimetry; D003353:Corynebacterium diphtheriae / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D009532:Nickel / Q000494:pharmacology*; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D008024:Ligands; D006639:Histidine; D009532:Nickel; D003545:Cysteine