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

Abstract

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 spore-specific peptidoglycan modification, muramic-∂-lactam (MAL), which is specifically recognized by cortex lytic enzymes. In C. difficile, MAL production depends on the CwlD amidase and its binding partner, the GerS lipoprotein. To gain insight into how GerS regulates CwlD activity, we solved the crystal structure of the CwlD:GerS complex. In this structure, a GerS homodimer is bound to two CwlD monomers such that the CwlD active sites are exposed. Although CwlD structurally resembles amidase_3 family members, we found that CwlD does not bind Zn2+ stably on its own, unlike previously characterized amidase_3 enzymes. Instead, GerS binding to CwlD promotes CwlD 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.

Metal cofactor stabilization by a partner protein is a widespread strategy employed for amidase activation

“…The LytH amidase domain consists of a twisted six-stranded beta-sheet surrounded by six alpha helices. The fold is highly conserved with solved structures of other proteins in the amidase_3 family ( 23 – 32 ; Protein Data Bank [PDB] ID codes 1JWQ, 3CZX, and 4RN7; PFAM ID PF01520). In our crystal structure, we observed a metal ion in the active site (initially assumed to be zinc, but see below) with an octahedral coordination sphere made up of four amino acid side chains (H128, E145, H193, and D195) and two water molecules ( Fig.…”

“…Recently, the Clostridioides difficile lipoprotein GerS was also found to stabilize zinc binding in the amidase CwlD to promote activity ( 32 ). Moreover, as with ActH and LytH, metal cofactor binding was found to be important for stable association between GerS and CwlD.…”

Mechanistic Insights into the Activities of Major Families of Enzymes in Bacterial Peptidoglycan Assembly and Breakdown

“…Similarly, in C. difficile , amidase CwlD activity is controlled by its allosteric activator GerS, which also facilitates Zn 2+ binding and catalysis, where the N ‐terminus of GerS was shown to directly participate in Zn 2+ stabilisation . [204] …”

Reconstituting Spore Cortex Peptidoglycan Biosynthesis Reveals a Deacetylase That Catalyzes Transamidation

“…Analogous experiments conducted with the CwlD and PdaA variants from C. difficile , Cd CwlD and Cd PdaA1, respectively, produced similar results, although the amidase activity of Cd CwlD was weak (Figure S4). This is presumably because our reaction mixtures did not contain GerS, an activator of Cd CwlD required for germination in this organism. , Nevertheless, Cd PdaA1 activity was robust, with all product B converted to C or lactam (Figure S4).…”

“…CwlD and PdaA are both metalloenzymes that contain a divalent cation cofactor, often Zn 2+ , used to promote the nucleophilicity of a water molecule (Figure S6). , Their reactions can therefore be quenched by addition of excess chelator such as EDTA (Figure S7). , To assess activity over time, we treated linear peptidoglycan with Bs CwlD to generate polymer enriched in peptide-cleaved product B and incubated the resulting material with Bs PdaA or Cd PdaA1.…”