Exploring protein secretion-modulating and structural features of pro-peptides from S8 class subtilisin-like serine proteases in Bacillus subtilis

One of the most common post-translational modifications occurring in bacteria is proteolytic cleavage. Particularly, in secretory proteins this cleavage is observed for specific protein segments, named pro-peptides, which are frequently located between the signal peptide and the mature protein. Pro-peptides are known to serve two main functions, namely a chaperone function in protein folding and an enzyme-inhibiting function. To date, the contribution of pro-peptides to protein secretion is not fully understood. Therefore, the present study was aimed at assessing possible roles of the N-terminal pro-peptides from the S8 class subtilisin-like serine proteases Bpr, Vpr and WprA of Bacillus subtilis in protein secretion. To this end, the signal peptides of these proteases were fused to the alkaline phosphatase PhoA of Escherichia coli with or without the cognate pro-peptides. Subsequently, secretion of the PhoA protein and its activity were assessed in the B. subtilis type strain 168 or the genome-reduced strain IIG-Bs27-39 and correlated to the respective pro-peptide structure as predicted by AlphaFold. Altogether, the obtained results suggest that the contributions of the investigated pro-peptides to protein secretion in B. subtilis are related to a combination of their functions in protein folding, their own folding state, and the applied strain.