Mutational analysis of the pro-peptide of a marine intracellular subtilisin protease supports its role in inhibition
AuthorBjerga, Gro Elin Kjæreng; Larsen, Øivind; ARSIN, Hasan; Williamson, Adele Kim; Garcia-Moyano, Antonio; Leiros, Ingar; Puntervoll, Pål
Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E. coli, and is active in presence of calcium, which is thought to have a role in minor, but essential, structural rearrangements needed for catalytic activity. The ISP operates at alkaline pH and at moderate temperatures, and has a corresponding melting temperature around 60 °C. The high‐resolution 3‐dimensional structure reported here, represents an ISP with an intact catalytic triad albeit in a configuration with an inhibitory pro‐peptide bound. The pro‐peptide is removed in other homologs, but the removal of the pro‐peptide from the i>Planococcus sp. AW02J18 ISP appears to be different, and possibly involves several steps. A first processing step is described here as the removal of 2 immediate N‐terminal residues. Furthermore, the pro‐peptide contains a conserved LIPY/F‐motif, which was found to be involved in inhibition of the catalytic activity.
This is the pre-peer reviewed version of the following article: Bjerga, G.E.K., Larsen, Ø., Arsın, H., Williamson, A., García-Moyano, A., Leiros, I. & Puntervoll, P. (2018). Mutational analysis of the pro-peptide of a marine intracellular subtilisin protease supports its role in inhibition. Proteins: Structure, Function, and Bioinformatics, which has been published in final form at https://doi.org/10.1002/prot.25528. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.