Strand-swapping in the LolA-like protein GerS promotes the allosteric activation of a bacterial amidase in Clostridioides difficile
GerS is a key lipoprotein regulator of Clostridioides difficile spore germination that shares structural, but not sequence, similarity to LolA in Gram-negative bacteria. GerS and LolA have vastly different biological roles: LolA functions in the trafficking of lipoproteins across the periplasmic space to the outer membrane of Gram-negative bacteria, while GerS allosterically activates the germination-specific amidase CwlD in endospore-forming Gram-positive bacteria. Here, we explore the diversity of LolA-like proteins across bacteria and reveal that GerS homodimer formation is a conserved feature of Peptostreptococcaceae family orthologs. Since dimerization of C. difficile GerS occurs via strand-swapping, we sought to investigate the functional importance of its strand swap by identifying mutations that alter the GerS dimer:monomer equilibrium. Our analyses reveal that GerSK71R and GerSN74L substitutions in the strand-swap hinge of GerS stabilize the homodimer, while a GerST72P substitution promotes monomer formation. Conversely, substituting the highly conserved proline at the equivalent site in LolA for threonine converts LolA from a monomer to a homodimer. Since our data indicate that destabilizing the GerS dimer impairs both GerS:CwlD binding in vitro and CwlD function in C. difficile, strand-swapped dimer formation in LolA-like proteins enhances their affinity for their binding partners. These data are consistent with analyses of equivalent LolA mutants in E. coli and thus provide new insight into the evolution and specialization of this intriguing group of proteins across the bacterial domain.