Structural Basis of Response Regulator Inhibition by a Bacterial Anti-Activator Protein

Abstract

Author SummaryUpon phosphorylation, bacterial proteins called response regulators bind to DNA promoters and activate or repress transcription. These response regulators are themselves regulated by anti-activator proteins, which can control response regulator activity without altering their phosphorylation state. We have determined the X-ray crystal structure of the anti-activator RapF complexed with the DNA-binding domain of the response regulator ComA. Our structure-function studies show that RapF disrupts the binding of ComA to DNA using a two-pronged mechanism. First, a RapF surface mimics DNA, and this DNA-like surface binds to nearly all of the ComA DNA-binding residues, thus blocking ComA's interaction with DNA. Second, RapF inhibits ComA dimerization. RapF is also regulated by the PhrF peptide; we find that the RapF-ComA interaction surface is distant from the proposed PhrF binding site. Furthermore, we found that RapF undergoes a conformational change upon binding to PhrF, which likely causes its dissociation from ComA. From these observations, we conclude that PhrF binding to RapF allosterically triggers its dissociation from ComA. Finally, we compared the RapF/ComA DNA-binding domain complex structure with the structure of another response regulator, Spo0F, complexed with the phosphatase RapH. This reveals that while RapF and RapH are structurally similar, they have evolved distinct, non-overlapping surfaces to interact with their different cellular targets.