IDENTIFICATION OF THE PRIMARY METAL ION-ACTIVATION SITES OF THE DIPHTHERIA, TOX REPRESSOR BY X-RAY CRYSTALLOGRAPHY AND SITE-DIRECTED MUTATIONAL ANALYSIS

The diphtheria fox repressor, DtxR, is a 226 amino acid transition met al ion-activated regulatory protein that controls the expression of di phtheria toxin in toxigenic Corynebacterium diphtheriae. The previousl y solved three-dimensional DtxR structures have identified two potenti al metal ion binding sites which may play a role in the activation of DNA binding by the repressor. We have used both X-ray crystallographic and site-directed mutational analysis of DtxR(C102D)-Ni2+ complexes a nd DtxR to identify the metal ion-binding site which results in the ac tivation of the repressor. We demonstrate that DtxR contains both a pr imary and an ancillary metal ion binding site. The primary site functi ons directly in the activation of DNA binding. In contrast, the ancill ary site contributes weakly, if at all, to activation.