Abstract
Author SummaryA phosphorelay signal transduction pathway regulates sporulation in numerous Bacillus species including the genetic model organism, B. subtilis, and the causative agent of anthrax, B. anthracis. Histidine kinases initiate the flow of phosphoryl groups along the phosphorelay pathway, which then shuttles them to a downstream response-regulator transcription factor called Spo0A. Ultimately, sporulation is governed by the cellular concentration of phosphorylated Spo0A. In numerous Bacillus species, Rap phosphatases function in opposition to the histidine kinases, inhibiting Spo0A activation by dephosphorylating an intermediate pathway protein called Spo0F. Here we present the structure of a Rap protein, RapH, in complex with Spo0F, as determined by X-ray crystallography. The RapH–Spo0F structure, along with biochemical and genetic studies, reveals the mechanism of Rap-protein-mediated Spo0F dephosphorylation. We used information gleaned from our structure–function analysis, first, to assign Spo0F phosphatase activity to an uncharacterized Rap protein, RapJ, on the basis of sequence alone, and, second, to engineer Spo0F phosphatase activity de novo into a non-phosphatase Rap protein, RapF. We found that in addition to dephosphorylating Spo0F, Rap proteins can inhibit the sporulation phosphorelay by sterically blocking the transfer of phosphoryl groups to and from Spo0F. Ultimately, new classes of drugs might be developed that disrupt the flow of phosphoryl groups along phosphotransfer signaling pathways by mimicking the antagonistic effects of Rap proteins on response regulators.
Highlights
B. subtilis spore development is regulated by a phosphorelay signal transduction pathway that is the prototype for all phosphorelay signal transduction pathways in bacteria (Figure 1; [1])
Overall RapH-Spo0F Crystal Structure To determine the mechanistic basis of Rap phosphatase activity, we crystallized B. subtilis RapH in complex with its target response regulator Spo0F
After 30 min only 16% of Spo0F,P remained in the RapF-H50L reaction while 54% remained in the wild-type RapF control (Figure 8C). These results, along with our structural analysis, reveal the mechanistic basis underlying the inability of wild-type RapF to dephosphorylate Spo0F and confirm that we have identified the biologically important Rap protein surface and many of the structural determinants required for Rap phosphatase activity
Summary
B. subtilis spore development is regulated by a phosphorelay signal transduction pathway that is the prototype for all phosphorelay signal transduction pathways in bacteria (Figure 1; [1]). The B. subtilis sporulation signal transduction pathway is initiated by five histidine kinases—KinA, KinB, KinC, KinD, and KinE—that autophosphorylate in response to unknown environmental and physiological cues coincident with nutritional starvation (Figure 1, left panel; [2]). Signaling converges on the centrally important intermediate response regulator Spo0F, which autophosphorylates using phosphohistidines in a sporulation histidine kinase as a phosphoryl donor. Spo0F phosphorylates the histidine phosphotransfer (HPt) protein Spo0B, which in the final pathway step relays phosphoryl groups to Spo0A. At least 520 B. subtilis genes are significantly upregulated or downregulated as a result of Spo0A phosphorylation [4]
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