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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.

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.

SUBMITTER: Duval V

PROVIDER: S-EPMC3719538 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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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.

Duval Valérie V McMurry Laura M LM Foster Kimberly K Head James F JF Levy Stuart B SB

Journal of bacteriology 20130517 15

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 s ...[more]

PMID: 23687277

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Project description:BackgroundBacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism.ResultsHere we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 Å resolution) and citrate-bound (Form-II, 1.90 Å resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K(m) in the order of acetate > propionate > formate. Further, the Km for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg(2+) could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn(2+) and Co(2+)), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic βββαβαβα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes.ConclusionsThe biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

Dataset Information

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

Publications

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

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