The src homology 3-like domain of the diphtheria toxin repressor (DtxR) modulates repressor activation through interaction with the ancillary metal ion-binding site.
The diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor that acts as a global regulatory element in the control of iron-sensitive genes in Corynebacterium diphtheriae. We recently described (L. Sun, J. C. vanderSpek, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 95:14985-14990, 1998) the isolation and in vivo characterization of a hyperactive mutant of DtxR, DtxR(E175K), that appeared to be constitutively active. We demonstrate here that while DtxR(E175K) remains active in vivo in the presence of 300 micro M 2,2'dipyridyl, the purified repressor is, in fact, dependent upon low levels of transition metal ion to transit from the inactive apo form to the active metal ion-bound form of the repressor. Binding studies using 8-anilino-1-naphthalenesulfonic acid suggest that the E175K mutation stabilizes an intermediate of the molten-globule form of the repressor, increasing exposure of hydrophobic residues to solvent. We demonstrate that the hyperactive DtxR(E175K) phenotype is dependent upon an intact ancillary metal ion-binding site (site 1) of the repressor. These observations support the hypothesis that metal ion binding in the ancillary site facilitates the conversion of the inactive apo-repressor to its active, operator-binding conformation. Furthermore, these results support the hypothesis that the C-terminal src homology 3-like domain of DtxR plays an active role in the modulation of repressor activity.
Love
John F
JF
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
VanderSpek
Johanna C
JC
Murphy
John R
JR
D019943:Amino Acid Substitution; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002412:Cations / Q000378:metabolism; D003353:Corynebacterium diphtheriae / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008670:Metals / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D002412:Cations; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D005456:Fluorescent Dyes; D008670:Metals; D011994:Recombinant Proteins; D012097:Repressor Proteins; C027132:1-anilino-8-naphthalenesulfonate