A lipoprotein allosterically activates the CwID amidase during Clostridioides difficile spore formation

Spore-forming pathogens like Clostridioides difficile depend on germination to initiate infection. During gemination, spores must degrade their cortex layer, which is a thick, protective layer of modified peptidoglycan. Cortex degradation depends on the presence of the sporespecific peptidoglycan modification, muramic-partial derivative-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwID amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwID activity, we solved the crystal structure of the CwID:GerS complex. In this structure, a GerS homodimer is bound to two CwID monomers such that the CwID active sites are exposed. Although CwID structurally resembles amidase_3 family members, we found that CwID does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwID promotes CwID binding to Zn2+, which is required for its catalytic mechanism. Thus, in determining the first structure of an amidase bound to its regulator, we reveal stabilization of Zn2+ co-factor binding as a novel mechanism for regulating bacterial amidase activity. Our results further suggest that allosteric regulation by binding partners may be a more widespread mode for regulating bacterial amidase activity than previously thought.

Automated summary

Spore-forming pathogens such as Clostridioides difficile rely on germination to cause infection, where the production of malamycin (MAL) is crucial for cortex degradation. The binding of GerS lipoprotein to CwID amidase not only promotes the stability of Zn2+ co-factor binding, but also reveals a novel mechanism for regulating bacterial amidase activity. This study suggests that allosteric regulation by binding partners may be more common in bacterial amidase regulation than previously understood.