Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein

Guangshun Wang, Gregory P. Wylie, Pamela D. Twigg, Donald L.D. Caspar, John R. Murphy, Timothy M. Logan

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Abstract

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C- terminal domain, consisting of residues N130-L226 plus a 13-residue N- terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five β- strands and three helices arranged into a partially orthogonal, two-sheet β- barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in β-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x- ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.

Original languageEnglish (US)
Pages (from-to)6119-6124
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number11
DOIs
StatePublished - May 25 1999

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