Inhibitory proteins of <i>Bacillus subtilis</i> interact within the membrane to block intramembrane protease activity

Intramembrane proteases (IPs) are crucial for diverse signaling pathways, including some that regulate the virulence of pathogenic bacteria. A better understanding of mechanisms controlling IP activity is necessary to guide therapeutic development. <i>Bacillus subtilis</i> SpoIVFB is an IP with two natural inhibitory proteins, BofA and SpoIVFA. These proteins form a complex with SpoIVFB and prevent it from cleaving Pro-σ^K during endosporulation. We investigated proximity between BofA and SpoIVFA in the SpoIVFB inhibition complex using <i>in vivo</i> disulfide crosslinking in <i>Escherichia coli</i>. We discovered that two parts of the BofA C-terminal region are proximal to the SpoIVFA transmembrane segment (TMS). Our results support predictions that the BofA C-terminal region adopts an unusual structure within the membrane and interacts with the SpoIVFA TMS to block SpoIVFB cleavage of Pro-σ^K. Endospore-forming <i>Bacilli</i>, including several pathogenic species, encode BofA and SpoIVFA orthologs, which likely interact in a similar fashion to inhibit their SpoIVFB ortholog. It may be possible to control SpoIVFB activity and hence endosporulation with modulators that target the interaction between BofA and SpoIVFA.IMPORTANCERegulated intramembrane proteolysis (RIP) pathways govern important processes in all three domains of life. A key component of RIP pathways is an intramembrane protease (IP), which cleaves one or more substrates within a membrane. Developing modulators of IPs has been challenging, particularly for metallo-IPs. Bacterial metallo-IPs function in RIP pathways that control the virulence of many pathogens. SpoIVFB is a metallo-IP necessary for endosporulation of <i>Bacillus subtilis</i>. Endosporulation enhances the survival of pathogenic <i>Bacilli</i>, which encode orthologs of SpoIVFB and its natural inhibitory proteins BofA and SpoIVFA. Here, we present the first experimental evidence for contacts between BofA and SpoIVFA within the membrane-embedded SpoIVFB inhibition complex, providing foundations for a deeper understanding of mechanisms controlling metallo-IP activity.