Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

Abstract

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.

Structural Basis of a Physical Blockage Mechanism for the Interaction of Response Regulator PmrA with Connector Protein PmrD from Klebsiella pneumoniae

“…Several crystal structures of receiver domains of response regulators have been solved (43)(44)(45)(46), but little structural information is available regarding how they interact with each other in multiprotein complexes. As well, knowledge of the structure of the receiver domain in complex with the connector protein is limited.…”

Novel antibacterial compounds specifically targeting the essential WalR response regulator

“…4,6,33 A mode of action B. subtilis and S. aureus WalRs, which were overproduced and purified from E. coli, revealed both monomeric and dimeric forms (Figure 6a-1, 2), whereas ArcA and OmpR formed the monomer as previously reported. [35][36][37] After WalR was treated with walrycin A at 30 1C for 60 min followed by size-exclusion chromatography, a decrease in the concentration of WalR monomer and a concomitant increase in the dimeric form (Figure 6a-1, 2) were observed. This increase in the proportion of dimeric protein was not observed when purified ArcA or OmpR was incubated with walrycin A (Figure 6a-3, 4).…”

Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

“…However, in vitro phosphorylation experiments with PleD resulted in an exiguous increase of DGC activity, possibly due to suboptimal assay conditions or to low stability of the phosphorylated form (4). For this reason we tested activation of PleD by beryllium fluoride (BeF 3 ) a molecular mimic of a phosphoryl group that has been widely used for biochemical and structural studies of bacterial response regulators (12)(13)(14)(15)(16)(17)(18). As shown in Fig.…”