Biophysical investigations into mechanisms for regulation of diphtheria toxin repressor activity

Diphtheria toxin repressor protein (DtxR) functions as a negative regulator of iron-sensitive genes in Corynebacterium diphtheriae, binding to DNA as a dimer in the presence of divalent metal cations. While crystallographic studies have provided structural information about the dimer in the apo- and holo-forms, no information is available about the atomic structure of the monomer. Understanding how metal binding regulates the activity of the diphtheria toxin repressor protein (DtxR) requires information about the structure in solution. We have prepared a DtxR mutant construct with three additional N-terminal residues, Gly-Ser-His-DtxR(Cys-102 → Asp), that retains metal-binding capabilities, but remains monomeric in solution and does not bind DNA under conditions that effect dimerization and DNA binding in the functional DtxR(Cys-102 → Asp) construct. The mutant construct gsh-DtxR(C102D) crystallizes as a dimer with an overall structure like that of wild-type DtxR and DtxR(C102D) but, in light of its very different solution properties, raises questions about conformational bias imposed by the crystallization conditions.; Solution nuclear magnetic resonance spectra of diphtheria toxin repressor demonstrate that while the C-terminal domain (residues 149–226) of the protein is well-ordered, the N-terminal domain (residues 1–13) exhibits conformational flexibility and exists as an ensemble of structural substates. Fluorescence binding assays with 1-anilino naphthalene-8-sulfonic acid (ANS) and circular dichroism spectroscopy confirm that the N-terminal domain of the apo-protein is a molten globule-type structure in solution with a high degree of helical secondary structure, but an undefined tertiary structure. Binding of divalent metal cations induces a substantial conformational reorganization, as evidenced by changes in the NMR spectra and ANS binding. The evident disorder to order transition upon binding of metal in solution is in contrast to the minor conformational changes seen comparing apo- and holo-DtxR crystal structures. Disordered to ordered folding appears to be a general mechanism for regulating specific recognition in protein action and this mechanism provides a plausible explanation for how metal binding controls the DtxR repressor activity.