Disordered to ordered folding in the regulation of diphtheria toxin repressor activity

Understanding how metal binding regulates the activity of the diphtheria to xin repressor protein (DtxR) requires information about the structure in so lution. We have prepared a DtxR mutant construct with three additional N-te rminal residues, Gly-Ser-His-DtxR(Cys-102 --> Asp), that retains metal-bind ing capabilities, but remains monomeric in solution and does not bind DNA u nder conditions that effect dimerization and DNA binding in the functional DtxR(Cys-102 --> Asp) construct. Although the interaction properties of thi s inactive mutant in solution are very different from that of active repres sors, crystallization imposes the same dimeric structure as observed in all crystal forms of the active repressor with and without bound metal. Our so lution NMR analyses of active and inactive metal-free diphtheria toxin repr essors demonstrate that whereas the C-terminal one-third of the protein is well ordered, the N-terminal two-thirds exhibits conformational flexibility and exists as an ensemble of structural substates with undefined tertiary structure. Fluorescence binding assays with 1-anilino naphthalene-8-sulfoni c acid (ANS) confirm that the highly alpha -helical N-terminal two-thirds o f the apoprotein is molten globule-like in solution. Binding of divalent me tal cations induces a substantial conformational reorganization to a more o rdered state, as evidenced by changes in the NMR spectra and ANS binding. T he evident disorder to order transition upon binding of metal in solution i s in contrast to the minor conformational changes seen comparing apo- and h olo-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 cont rols the DtxR repressor activity.