ABSTRACT
Bacterial signal transduction systems can be viewed as an entity of multi-sensory and output domains, whereas the functions of response regulators play a pivotal role in the complex network interactions. One crucial property among response regulators functions is their oligomerization and subsequent binding to DNA. The AtoS-AtoC two component system, functionally modulated by various agents, influences fundamental cellular processes such as short-chain fatty acid catabolism and poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli. Among the already reported characteristic properties, AtoC binds to a specific site, a palindromic repeat of 20 nucleotides within the atoDAEB promoter. Since experimental structures of AtoC or its complex with DNA are not yet available, an almost complete homology model of AtoC and of its putative entity as a dimer is constructed for this study, as well as a model of its binding to its target DNA sequence. The latter is associated with large conformational changes, as shown by molecular dynamics simulations. Subsequent biochemical study, including cross-linking via chemical agents, revealed the ability of AtoC to form oligomers in vitro.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Models, Molecular , Protein Multimerization , Amino Acid Sequence , Amino Acids/metabolism , Base Sequence , Binding Sites , Cross-Linking Reagents/pharmacology , DNA/genetics , Molecular Sequence Data , Protein Binding/drug effects , Protein Multimerization/drug effects , Protein Structure, Quaternary , Protein Structure, Secondary , Reproducibility of Results , Static ElectricityABSTRACT
The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.
