Structural Basis of Rap Phosphatase Inhibition by Phr Peptides

Abstract

A structural and functional study shows the molecular mechanism of Rap protein inhibition by Phr signaling peptides, providing new insights into peptide recognition and discrimination in quorum sensing.

Abstract

Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change.

Author Summary

In 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.