Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains

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.     

Pivotal roles for the receiver domain in the mechanism of action of the response regulator RamR of Streptomyces coelicolor

The response regulator RamR activates expression of the ramCSAB operon, the source of the morphogenetic peptide SapB, and is therefore important for morphogenesis of the bacterium Streptomyces coelicolor. Like most response regulators, RamR consists of an amino-terminal receiver domain and a carboxy-terminal DNA binding domain. Four of five highly conserved active site residues known to be important in other response regulators are present in RamR: D12, D56 (the predicted site of phosphorylation), T84 and K105. Here, we show that in spite of this, RamR did not demonstrate an ability to autophosphorylate in vitro in the presence of small molecule phosphodonors. The unphosphorylated protein behaved as a dimer and bound cooperatively to three sites in the ramC promoter, one with very high affinity and two with lower affinity. On its own, the RamR DNA binding domain could not bind DNA but was able to interfere with the action of full length RamR in a manner suggesting direct protein-protein contact. Surprisingly, substitution of residues D12 or T84 had no effect on RamR function in vivo. In contrast, D56A and K105A substitutions caused defects in both dimer formation and DNA binding while the more conservative substitution, D56N permitted dimer formation but not DNA binding. L102 in RamR corresponds to a well-conserved tyrosine (or aromatic) residue that is important for function in the other response regulators. While a L102Y variant, which introduced the aromatic side-chain usually found at this position, functioned normally, L102A and L102W substitutions blocked RamR function in vivo. We show that these substitutions specifically impaired cooperative DNA binding by RamR at the lower affinity recognition sequences. The biochemical properties of RamR therefore differ markedly from those of other well-characterized response regulators.     

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.