The diphtheria toxin repressor, DtxR has been cloned from genomic libraries of Corynebacterium diphtheria. DtxR folds into a N-terminal (M1-G136) and C-terminal (R161-L226) domain structure which are connected by a random coil linker (E137-N160). The N-terminal domain of DtxR contains the helix-turn-helix DNA binding motif, the primarily and ancillary metal ion-binding domain, and a protein-protein interaction region. Until recently, the structure of the C-terminal domain of DtxR (Qiu et al., 1997) and NMR spectroscopy of the C-terminal peptide DtxR (C102D) bind to opposite sides of the diphtheria tox operator following a metal-ion triggered subunit """"""""caliper-like"""""""" movement which aligns the HTH motif in the major groove and a helix-to-coil transition of the N- terminal six amino acids )White et al., 1998). Apo-DtxR is an inactive monomeric form; however, upon addition of activating metal ions, DtxR undergoes a conformational change to an active dimeric structure. During the last grant period we have developed an extremely powerful positive genetic selection system (PSDT) that links chloramphenicol resistance to a functional DtxR:tox operator genetic circuit. Using the PSDT system we have isolated the first self-activated iron-independent mutants of DtxR. The simplest mutant in this new class, DtxR (E175K) carries a single point mutation in its SH3-like domain. Given the apparent flexibility of this C-terminal SH3-like domain we propose that the epsilon-amino moiety of K175 in this mutant is able to insert into the primary metal ion-binding site and serves as a surrogate for iron in the activation of repressor activity. Based upon this hypothesis, we have screened a peptide library for peptides capable of activating wild type DtxR. This search has yielded the first iron-mimetic peptides capable of activating wild type DtxR. This search has yielded the first iron-mimetic peptides capable of activating wild type DtxR repressor activity in the absence of iron. The long term goals of this proposal are focused on the elucidation of the molecular events which modulate the conversion of inactive apo-DtxR to its active dimeric form, and the mechanism of peptide-mediated activation of repressor activity in the absence of iron. We anticipate that iron mimetic activations of the DtxR family of repressors may be prototypes of a new class of """"""""antibiotic"""""""" that selectivity repress iron-sensitive gene expression and thereby attenuate virulence.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021628-18
Application #
6627967
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Klein, David L
Project Start
1984-05-01
Project End
2005-01-31
Budget Start
2003-02-01
Budget End
2004-01-31
Support Year
18
Fiscal Year
2003
Total Cost
$311,406
Indirect Cost
Name
Boston Medical Center
Department
Type
DUNS #
005492160
City
Boston
State
MA
Country
United States
Zip Code
02118
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