Disordered to ordered folding in the regulation of diphtheria toxin repressor activity.
نویسندگان
چکیده
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. Although the interaction properties of this inactive mutant in solution are very different from that of active repressors, crystallization imposes the same dimeric structure as observed in all crystal forms of the active repressor with and without bound metal. Our solution NMR analyses of active and inactive metal-free diphtheria toxin repressors 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-sulfonic acid (ANS) confirm that the highly alpha-helical N-terminal two-thirds of the apoprotein is molten globule-like in solution. Binding of divalent metal cations induces a substantial conformational reorganization to a more ordered state, 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.
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عنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 98 20 شماره
صفحات -
تاریخ انتشار 2001