Delineation of the Residues of Bacillus anthracis Zinc Uptake Regulator Protein Directly Involved in Its Interaction with Cognate DNA

Lay Summary

Based on the structural and mutational analysis, Leu51, Phe53, Arg58, and Phe63 residues of the HTH motif of Zur of B. anthracis were delineated as the DNA-binding hotspot of BaZur involved in the binding to the specific sequence (Zur box) in the promoter DNA of its regulon candidates. The other residues constituting the DNA-binding HTH motif, as predicted by homology modelling, could be involved in an assistive role in the DNA-protein interaction. Through sequence analysis, we can conclude that at least Arg58 is a highly conserved and a signature residue throughout the family of Zur proteins, which allows all the Zur subtypes to converge into a single group of proteins.

Abstract

Zinc uptake regulator (Zur) is a negative transcriptional regulator of bacteria that belongs to the FUR superfamily of proteins and regulates zinc (Zn) homeostasis under extreme Zn conditions. The Zur protein of Bacillus anthracis (BaZur) was though characterized previously, but the residues of this transcriptional regulator, crucial for binding to the consensus Zur box in the cognate DNA, remain unexplored. In this study, we reveal the essential residues of the protein that govern the specific interaction with the cognate DNA, through mutational and binding studies. In silico predicted model of the BaZur protein with the promoter region of one of the regulon candidates was utilized to identify specific residues of the N-terminal domain (NTD), constituting the DNA-binding recognition helix. Our results suggest that two phenylalanine residues, a non-polar aliphatic leucine and a positively charged arginine residue of NTD, are predominantly involved in DNA binding of BaZur. Among these, the arginine residue (Arg58) is conserved among all the Zur proteins and the two Phe residues, namely Phe53 and Phe63, are conserved in the Zur proteins of Staphylococcus aureus and Listeria monocytogenes. Taken together, the current study represents an in-depth investigation into the key DNA-binding residues involved in the BaZur-DNA interaction.

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