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.
Highlights
Two-component signaling systems, consisting of a sensor histidine kinase and a response regulator transcription factor, are the principal mechanism of signal transduction in bacteria [1]
The Rap proteins are a family of auxiliary factors that have been most thoroughly studied in Bacillus subtilis, where they regulate two-component and phosphorelay signal transduction
RapF Structure RapF consists of two distinct domains, an N-terminal antiparallel 3-helix bundle and a C-terminal tetratricopeptide repeat (TPR) domain (Figure 2B)
Summary
Two-component signaling systems, consisting of a sensor histidine kinase and a response regulator transcription factor, are the principal mechanism of signal transduction in bacteria [1] Upon phosphorylation of their receiver (REC) domains, response regulators bind target DNA promoters and activate or repress transcription. A number of auxiliary factors have garnered significant attention because they function as anti-activators that inhibit response regulator function without affecting their phosphorylation state. These proteins inhibit the interaction of response regulators with their target promoters [2,3,4,5] or RNA polymerase [6,7]. Rap proteins influence diverse B. subtilis developmental and cellular differentiation processes including sporulation, genetic competence, and biofilm formation, as well as the production of secreted enzymes and the movement of a conjugative transposon [8,9]
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