Abstract
Anthracis Repressor (AntR) is a Mn(II) activated DNA binding protein that is involved in the regulation of Mn(II) homeostasis in Bacillus anthracis. AntR is a member of the Diphtheria Toxin Repressor (DtxR) family of proteins. These proteins function as sensors of intracellular Fe(II) or Mn(II) levels and effect the metal regulated expression of many genes, frequently including virulence related genes. Our studies on AntR focus on metal regulated activation of the protein. We have determined the Mn(II) binding stoichiometry, equilibrium binding constants, and associated kinetic rate constants in AntR using a variety of electron paramagnetic resonance methods. Two divalent manganese ions were observed to bind AntR with positive cooperativity and apparent dissociation constants of 210 ± 18 μM and 16.6 ± 1.0 μM. Binding rates were in the sub-millisecond range, and dissociation rates were characterized by rate constants 35.7 ± 12.1 s-1 and 0.115 ± 0.009 s-1. We probed the nature of the metal binding site with EPR for comparison with the crystal structures of homologous manganese transport regulator (MntR) from Bacillus subtilis. The spectra were not consistent with a binuclear Mn(II) cluster as seen in MntR structures. Gel filtration, continuous wave EPR, and Pulsed EPR methods were used to investigate possible structural changes in response to metal binding. We found that AntR is exclusively dimeric in absence of Mn(II). Double electron-electron resonance (DEER) was employed to measure spin-spin distance of strategically placed nitroxide spin labels in dimeric AntR. To realize the full potential of DEER, an analysis software with graphical user interface was developed. The data indicated the presence of multiple conformations for each spin label pair in apo-AntR. Metal binding had little effect on these conformations, except near the putative DNA-binding helixes, where metal binding sharpened the distribution of conformers, and decreased the distance between DNA binding regions of AntR dimer. We also showed that the AntR backbone dynamics change considerably upon metal binding. A structure model for AntR was built from homology to MntR, and the experimentally measured distances were simulated. This model only partially agreed with the DEER results, suggesting structural differences between AntR and MntR. These results allow us to develop a model for the Mn(II) induced activation of the repressor.
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