Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.
The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.
Tao
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Evans Department of Clinical Research, Boston University Medical Center Hospital, MA 02118, USA.
Zeng
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Murphy
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JR
D001059:Apoproteins / Q000187:drug effects / Q000378:metabolism*; D001426:Bacterial Proteins / Q000187:drug effects / Q000378:metabolism*; D001667:Binding, Competitive; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008345:Manganese / Q000494:pharmacology*; D008957:Models, Genetic; D009532:Nickel / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D001059:Apoproteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008345:Manganese; D009532:Nickel