Insights into AMS/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease

The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.This work was supported by grants from the National Institutes of Health (GM R37 058822 to WAV and GM079038 to SKN), and MINECO/FEDER (CTQ2015-70524-R and RYC-2013–14706 grants to GJO). NM acknowledges Universidad de La Rioja for a predoctoral fellowship. We thank Dr. William Kelly (AgResearch, New Zealand) for providing Lachnospiraceae bacterium C6A11, and Keith Brister and colleagues for facilitating data collection at LS-CAT (Argonne National Labs, IL)