Cell Signaling | Microbial Growth & Development | Protein Folding | Cell Signaling & Trafficking Structures | Experimental Biophysical Methods | Control of Gene Expression
Structural basis of response regulator dephosphorylation by Rap phosphatases
Vijay K Parashar, Nicolas Mirouze , David A Dubnau, Matthew B Neiditch*
*Corresponding author: Matthew B Neiditch
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA
F1000Posters 2012, 3: 74 (poster) [English]
Gordon Research Conference - Sensory Transduction in Microorganisms 2012, 15 - 20 Jan 2012, 14
New York Academy of Sciences 2012 - New York Structural Biology Discussion Group 7th Winter Meeting, 22 Jan 2012, 2
A 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.
The X-ray crystal structure of the response regulator phosphatase RapH in complex with one of its cellular targets, Spo0F, combined with extensive biochemical and genetic analysis of RapH mutants, reveals the mechanism of Rap-mediated phosphatase activity, along with many of the structural requirements for Rap phosphatase function.
The approach presented here that enabled us to assign Spo0F phosphatase function to RapJ, based only on its amino acid sequence, could be used to predict the target specificity of non-Spo0F phosphatase Rap proteins following the determination of structures of representative complexes of the Rap proteins bound to their targets. Furthermore, identifying the non-Spo0F phosphatase Rap protein residues important for target recognition will facilitate (1) the identification of Rap proteins whose sequences suggest that they may not recognize previously identified substrates, (2) the identification of new Rap protein targets, and (3) rational engineering of de novo Rap protein target specificity. The process of developing Rap proteins with engineered target specificity will not only provide valuable insight into the principles governing protein-protein interaction, but will also provide valuable reagents for studying bacterial signal transduction. Finally, insight gleaned from our structure-function analysis of RapH-Spo0F, along with additional studies of other Rap proteins in complex with their response regulators, could be used to develop new classes of drugs that disrupt the flow of information along bacterial phosphotransfer signaling pathways, by mimicking the antagonistic effects of Rap proteins on response regulators.
No relevant competing interests disclosed.
National Institutes of Health (NIH), R01AI081736
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