Regulatory function and mechanisms of the Rap-Phr cassettes
The regulatory function and mechanism of action of the Rap phosphatases and Phr peptides has been intensively studied. The known Rap phosphatases have been studied in diverse strains, finding an apparent redundancy in their function: most Rap phosphatases act upon Spo0F∼P, ComA∼P, or both . Furthermore, these investigations have revealed that certain Rap-Phr cassettes are encoded on plasmids, and that these regulatory modulators are a common feature of other members of theBacillus genus . Table 1 presents the known function of those Rap phosphatases that have been studied or reported independently, along with their possible action mechanism.
All known Rap phosphatases share a high sequence homology, however they regulate structurally distinct targets . Initial structural predictions of Rap phosphatases based on amino acid sequence suggested a two-domain architecture consisting of an N-terminal 3-helix bundle domain connected to a tetratricopeptide repeat (TPR) domain. This structure is strikingly different from other known bacterial phosphatases . TPR domains consist of 3 to 16 repeats of a degenerate 34 amino acid sequence motif, these repeats create a right-handed superhelix structure with an internal ligand-binding concave surface. TPR domains are known to function as protein-protein interaction domains . Rap proteins appear to possess 6 canonical TPR motifs distributed along most of their length, with a further non-canonical TPR motif separating TPR5 and TPR6 . Parasharet al. found that Rap proteins undergo a major conformational change in their N-terminal domain when complexed with their target proteins: the N-terminal 3-helix bundle is flipped and merged with the existing C-terminal TPR domains . Further comparison of the crystal structures of RapI, RapH and RapF (these last two in complex with their target proteins) revealed that these conformational changes can generate different interacting surfaces that block their target’s active site (in the case of Spo0F), or DNA-binding domain (in the case of ComA) .
The regulatory mechanism of the Phr peptides has also been structurally studied. Binding of Rap proteins to their cognate Phr peptides is mediated by their C-terminal TPR domains, and causes a pronounced rotation of the N-terminal 3-helix bundle; this creates two helix-turn-helix structures that pack against the existing C-teminal TPR domain. This rearrangement generates a compression along the whole TPR superhelical axis, which causes the loss of the ligand-binding concave surface normally present in Rap proteins. Furthermore, the Phr peptides can interact with the residues of multiple TPR repeats (up to six, in the case of RapF-PhrF complexes), leading to intramolecular interactions that stabilize the “closed” conformation of the Rap protein . These multi-TPR motif interactions confer a high specificity to Rap-Phr binding, with some Phr residues determining protein anchoring and orientation, and others mediating the interaction with the residues of the Rap protein. Gallego del Sol and Marina (2013) demonstrated that specific residues of RapF are required to bind its PhrF inhibitor, and that these residues are independent from the ability of RapF to bind to its target regulator ComA. The conservation of similar residues among Rap proteins, and additional experimental evidence from previous studies , suggest that this is a common Phr-binding mechanism for all Rap proteins.
Interestingly, a few known rap genes lack the concomitant gene for a specialized Phr peptide, but can be regulated by Phr peptides produced by other Rap-Phr cassettes (see SubtiWiki http://subtiwiki.uni-goettingen.de) . This is the case of RapB, which lacks a specialized Phr but is regulated by PhrC in vitro . Another example is rapJ , which is not followed by a phrgene. RapJ plays its regulatory role by dephosphorylating Spo0F∼P, and it binds to PhrC, forming a complex that is no longer able to interact with Spo0F∼P . Moreover, at least one Rap protein is known to be insensitive to regulation by its cognate Phr peptide. TherapP-phrP cassette is encoded in the pBS32 plasmid present in the undomesticated strain of B. subtilis NCIB 3610. RapP regulates biofilm formation, sporulation, and competence development by directly dephosphorylating Spo0F∼P , and by a ComA-dependent mechanism . However, RapP is not inhibited by PhrP, either when PhrP is overexpressedin vivo, or tested in vitro with exogenously added peptides derived from the C-terminal sequence of phrP . Conspicuously, RapP of B. subtilis NCIB 3610 shows an asparagine-to-threonine mutation at position 236 that is not present in the corresponding rapP alleles of other Bacillus strains. Omer Bendori et al . (2015) showed that this single amino acid substitution is responsible for the observed resistance of RapP to inhibition by PhrP, and that this inhibition could be restored by repairing the N236T mutation. Similarly, the plasmid encoded Rap63-Phr63 and Rap8-Phr8 modules synergistically moderate sporulation and biofilm formation of B. thuringiensis .
The structural insights from these studies suggest that there is a delicate balance between peptide-recognition specificity and regulatory plasticity in the Rap-Phr family. Studies that use various synthetic Phr peptides to investigate Rap-Phr interactions have found that, although usually one peptide shows strong affinity for a given Rap protein (normally the one coded in the same rap-phr cassette), other Phr peptides also show a partial ability to regulate the same Rap proteinin vitro and in vivo . These alternative Phr are usually synthetized comprising the last 5 or 6 residues of the C-terminal end of Phr pro-peptides . However, it is important to consider that these results have been observed using artificial laboratory conditions and, in particular, in vitro experiments include the testing of a very limited number of peptides at a time. In natural settings, a complex network of Phr peptide cross-talk and co-regulation might exist among the populations of Bacilli to modulate the function of Rap phosphatases.