Structural Studies of Bypass of Forespore Protein C from Bacillus Subtilis to Reveal Its Inhibitory Molecular Mechanism for SpoIVB

Abstract

Activation of pro-σK processing requires a signaling protease SpoIVB that is secreted from the forespore into the space between the two cells during sporulation in Bacillus subtilis. Bypass of forespore protein C (BofC) is an inhibitor preventing the autoproteolysis of SpoIVB, ensuring the factor σK operates regularly at the correct time during the sporulation. However, the regulatory mechanisms of BofC on pro-σK processing are still unclear, especially in the aspect of the interaction between BofC and SpoIVB. Herein, the recombinant BofC (rBofC) was expressed in the periplasm by the E. coli expression system, and crystal growth conditions were obtained and optimized. Further, the crystal structure of rBofC was determined by X-ray crystallography, which is nearly identical to the structures determined by NMR and predicted by AlphaFold. In addition, the modeled structure of the BofC–SpoIVB complex provides insights into the molecular mechanism by which domain 1 of BofC occupies the active site of the SpoIVB serine protease domain, leading to the inhibition of the catalytical activity of SpoIVB and prevention of the substrate of SpoIVB (SpoIVFA) from binding to the active site.