Bacterial sporulation and the SpoIV RIP pathway
Cellular differentiation is a process commonly associated with multi-cellular organisms and their development. However, differentiation into specialized cell types is also employed by a range of bacteria. One classic example is the endospore forming bacterium Bacillus subtilis that can differentiate into dormant and stress-resistant spores to survive harsh environmental conditions. Spore formation proceeds via an asymmetric cell division resulting in a small cell, the forespore, and a large mother cell. The final steps of spore formation are under control of the sigma factor sK, which is synthesized in the mother cell as an inactive, membrane-associated precursor protein pro-sK that is activated by regulated intramembrane proteolysis (RIP). In the specialized SpoIV RIP pathway, the I-CLiP intramembrane cleaving protease SpoIVFB (4FB) is embedded in the mother-cell membrane and is held inactive by two membrane proteins SpoIVFA (4FA) and BofA. Inhibition of 4FB is relieved by the signaling proteases SpoIVB (4B) and CtpB that are secreted into the intramembrane space and degrade the extracellular domain of the negative regulator 4FA. The 4FB membrane protease then liberates sK, which, in turn, activates many of the genes required to complete the sporulation program.
The signaling proteases 4B and CtpB belong to the widespread family of PDZ-proteases. Whereas 4B belongs to a small clan of bacterial serine proteases of unknown structure, CtpB is a member of the large family of C-terminal processing proteases that either serve a regulatory role when cutting the C-termini of specific precursor proteins or a housekeeping function when degrading incompletely synthesized proteins tagged by the SsrA system.
Our findings: 2013 (PDZ-gated protease tunnel of CtpB)
2013 CtpB composes the structural bottleneck of the SpoIV sporulation RIP pathway
Spore formation in Bacillus subtilis relies on a RIP pathway that synchronizes mother-cell and forespore development. To address the molecular basis of this SpoIV transmembrane signaling, we carried out a structure-function analysis of the activating protease CtpB. Crystal structures reflecting distinct functional states show that CtpB constitutes a ring-like protein scaffold penetrated by two narrow tunnels. Access to the proteolytic sites sequestered within these tunnels is controlled by PDZ domains that rearrange upon substrate binding. Accordingly, CtpB resembles a minimal version of a self-compartmentalizing protease regulated by a unique allosteric mechanism. Moreover, biochemical analysis of the PDZ-gated channel combined with sporulation assays revealed that activation of the SpoIV RIP pathway is induced by the concerted activity of the two signaling proteases CtpB and 4B.
As seen in one of the six CtpB crystal structures, the electron density of a co-purified ligand bound to the PDZ domain of CtpB is compatible with a Pro-Ala C-terminal motif. Intriguingly, a corresponding C-terminus is generated by the 4B protease. Though the processed 4FA C-terminus is not the preferred ligand of CtpB in vitro, protease and co-crystallization assays indicate that the weak affinity of the degradation tag is sufficient to activate CtpB. According to these data, the pre-digested 4FA substrate may need to reach a certain level to turn on CtpB activity. Such a mechanism would prevent the premature induction of the sporulation pathway and in parallel preclude the unspecific cleavage of non-native substrates carrying a similar degradation tag. In conclusion, we propose that 4B and CtpB act sequentially on 4FA with the 4B protease first removing the compact LytM domain and then, and only then, CtpB further cleaves 4FA removing the remaining linker region. According to our in vivo data, it is this linker segment that primarily inhibits the I-CLiP protease 4FB and must be removed to continue the SpoIV RIP pathway.
The discovered proteolytic mechanism is of broad relevance for cell-cell communication illustrating how different activation signals can be integrated into a single RIP pathway. Dependent on the origin of the signaling proteases such a mechanism could coordinate either different transcriptional programs within a single cell or, in case where the signaling proteases are secreted from two neighboring cells, link the RIP signal cascade to cell-cell communication coordinating for developmental programs, for example.