Abstract
Author SummaryIn microorganisms, two component signaling systems are widely used to sense and respond to environmental changes, including quorum-sensing of Phr oligopeptides. Although the minimal machinery required for these systems comprises a sensor histidine kinase and an effector response regulator (RR), ancillary proteins, termed “connectors,” capable of modulating the activity of this machinery, are emerging as additional players in this complex signaling process. Rap proteins are archetypal connectors, able to modulate the activity of RRs either by dephosphorylating them or by physically blocking them. Rap proteins are themselves in turn inhibited by specific Phr peptides, adding an extra level of complexity, but how a Rap protein is regulated by its cognate Phr peptide remains unknown. To answer this question, we solved the structure of RapF, a Rap family member that blocks RR ComA, alone and in the complex with its inhibitory peptide PhrF. Our structural and functional results reveal that PhrF blocks the RapF-ComA interaction by an allosteric mechanism since the PhrF-RapF interaction induces a conformational change that is propagated to the the ComA binding site, disrupting it and triggering the dissociation of ComA from RapF. Using sequence analysis guided by our structure, we pinpointed sets of residues responsible for peptide anchor and specificity, respectively, and were able to relax RapF-Phr specificity simply by changing a single residue. Knowledge of these key residues and the Rap inhibition mechanism opens up the possibility of re-engineering Rap proteins, and paves the way to reprogramming signaling pathways for biological and biotechnological applications.
Highlights
Bacteria communicate with each another and coordinate essential processes such as biofilm formation, sporulation, competence, virulence, or swarming motility in different ways
For some RNPP family members, it has been shown that the recognition and binding of signaling peptides is mediated by the tetratricopeptide repeat (TPR) [4,8,9,11]
The minimal machinery required for these systems comprises a sensor histidine kinase and an effector response regulator (RR), ancillary proteins, termed ‘‘connectors,’’ capable of modulating the activity of this machinery, are emerging as additional players in this complex signaling process
Summary
Bacteria communicate with each another and coordinate essential processes such as biofilm formation, sporulation, competence, virulence, or swarming motility in different ways. Quorum sensing is one of these mechanisms regulated by cell population density, and is mediated by self-generated extracellular signal molecules to allow the coordination of community-wide behaviors. Both Gram-negative and Gram-positive bacteria exploit quorumsensing signaling, generally through different messenger molecules. In the former, acyl-homoserine lactones are the predominant signals, whereas the quorum sensing in the latter relies on the secretion and recognition of oligopeptides. The RNPP family (named after its members: Rap/NprR/PlcR/ PrgX) of quorum-sensing proteins comprises Gram-positive regulators, which bind directly to their signaling peptide in the receiver cell [4,5]. The binding of signaling peptides to each RNPP representative seems to have dissimilar effects since PlcR and NrpR are activated, but Raps and PrgX are inhibited by their corresponding oligopeptides [8,12,14,15,16]
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