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.