SUMMARYEscherichia coli react to changes from aerobic to anaerobic conditions of growth using the ArcAArcB two-component signal transduction system. This system, in conjunction with other proteins, regulates the respiratory metabolic pathways in the organism. ArcA is a member of the OmpR/ PhoB subfamily of response regulator transcription factors that are known to regulate transcription by binding in tandem to target DNA direct repeats. It is still unclear in this subfamily how activation by phosphorylation of the regulatory domain of response regulators stimulates DNA binding by the effector domain and how dimerization and domain orientation, as well as intra-and intermolecular interactions, affect this process. In order to address these questions we have solved the crystal structure of the regulatory domain of ArcA in the presence and absence of the phosphoryl analog, BeF 3 − . In the crystal structures, the regulatory domain of ArcA forms a symmetric dimer mediated by the α4-β5-α5 face of the protein and involving a number of residues that are highly conserved in the OmpR/PhoB subfamily. It is hypothesized that members of this subfamily use a common mechanism of regulation by dimerization. Additional biophysical studies were employed to probe the oligomerization state of ArcA, as well as its individual domains, in solution. The solution studies show the propensity of the individual domains to associate into oligomers larger than the dimer observed for the intact protein, and suggest that the C-terminal DNA-binding domain also plays a role in oligomerization.
Bacterial response regulators are key regulatory proteins that function as the final elements of socalled two-component signaling systems. The activities of response regulators in vivo are modulated by phosphorylation that results from interactions between the response regulator and its cognate histidine protein kinase. The level of response regulator phosphorylation, which is regulated by intraor extracellular signals sensed by the histidine protein kinase, ultimately determines the output response that is initiated or carried out by the response regulator. We have recently hypothesized that in the OmpR/PhoB subfamily of response regulator transcription factors, this activation involves a common mechanism of dimerization using a set of highly conserved residues in the a4-b5-a5 face. Here we report the X-ray crystal structures of the regulatory domains of response regulators TorR (1.8 Å ), Ca 2+ -bound KdpE (2.0 Å ), and Mg 2+ /BeF 3 --bound KdpE (2.2 Å ), both members of the OmpR/ PhoB subfamily from Escherichia coli. Both regulatory domains form symmetric dimers in the asymmetric unit that involve the a4-b5-a5 face. As observed previously in other OmpR/PhoB response regulators, the dimer interfaces are mediated by highly conserved residues within this subfamily. These results provide further evidence that most all response regulators of the OmpR/ PhoB subfamily share a common mechanism of activation by dimerization.
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