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  • Mutational analysis of the multiple-antibiotic resistance regulator MarR reveals a ligand binding pocket at the interface between the dimerization and DNA binding domains.

Mutational analysis of the multiple-antibiotic resistance regulator MarR reveals a ligand binding pocket at the interface between the dimerization and DNA binding domains.

Journal of bacteriology (2013-05-21)
Valérie Duval, Laura M McMurry, Kimberly Foster, James F Head, Stuart B Levy
ABSTRACT

The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.

MATERIALS
Product Number
Brand
Product Description

Supelco
2,4-Dinitrophenol solution, certified reference material, 5000 μg/mL in methanol
Sigma-Aldrich
Sodium salicylate, puriss. p.a., ≥99.5% (NT)
Sigma-Aldrich
Sodium salicylate, tested according to Ph. Eur.
Sigma-Aldrich
Sodium salicylate, ReagentPlus®, ≥99.5%
Sigma-Aldrich
Sodium salicylate, meets USP testing specifications
Sigma-Aldrich
2,4-Dinitrophenol, moistened with water, ≥98.0%
Sigma-Aldrich
Sodium salicylate, puriss. p.a., reag. Ph. Eur., 99.5-101.0% (calc. to the dried substance)
Supelco
2,4-Dinitrophenol, PESTANAL®, analytical standard