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

Abstract

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.

Precursor peptide-targeted mining of more than one hundred thousand genomes expands the lanthipeptide natural product family

“…Examining these sequence motifs also reveals that in addition to the long-recognized FxLD sequence motif in the leader peptides of class I LanAs [36], a number of class I LanAs from Bacteroidetes have a LxLxKx 5 L motif instead. Many of the leader peptides that contain this motif end with a Gly-Gly sequence, and a C39-family Cys protease that removes leader peptides at GG sites [37,38] is often encoded in the corresponding clusters. This GG leader motif has previously only been observed in class II [39] and III LanAs [40].…”

A microcin processing peptidase‐like protein of the cyanobacterium Synechococcus elongatus is essential for secretion of biofilm‐promoting proteins

“…Studies of heterotrophic bacteria established a model in which the GG‐motif leader peptide is cleaved off during transport by the N‐terminal domain of a transporter component, which belongs to the Peptidase C39 protein family (Michiels et al, ; Bobeica et al, ). Mutation of the conserved cysteine of the peptidase domain of the protein encoded by Synpcc7942_1133 of S. elongatus (denoted PteB for Peptidase transporter enabling Biofilm), which belongs to the C39 family, indicated its requirement for biofilm development and for proper secretion of the EbfG small proteins (Parnasa et al, ).…”

Characterization of a Dehydratase and Methyltransferase in the Biosynthesis of Ribosomally Synthesized and Post‐translationally Modified Peptides in Lachnospiraceae

“…The leader peptides of all nine LahA peptides are conserved and, like the prochlorosins, are members of the Nif11‐like protein family ending with a double Gly motif (Figure C). Our previous study demonstrated that the LahT150 protease domain recognized this motif for all nine substrates . In contrast, the sequences of the corresponding core peptides exhibit a high degree of sequence divergence and five of the nine peptides distinctly lack Cys residues (Figure B).…”

“…Recently, we reported a highly substrate tolerant protease domain of the bifunctional transporter LahT encoded in a putative lanthipeptide biosynthetic gene cluster ( lah ) in Lachnospiraceae bacterium C6A11. This domain called LahT150 efficiently removes leader peptides from a large number of double glycine motif‐containing peptides . LahT is a member of the family of ABC‐transporter maturation and secretion (AMS) proteins (also called peptidase‐containing ATP‐binding transporters, PCAT) .…”

“…The lah biosynthetic gene cluster (BGC) in the genome of L. bacterium C6A11 encodes an intriguing compilation of RiPP PTM enzymes (Figure ). We identified the lah BGC by BLAST query searching for examples of BGCs that contain multiple precursor peptides based on the flavecin biosynthetic pathway . The lah cluster is unique in that it not only encodes at least seven precursor peptides with diverse core peptides, but also has a highly diverse set of PTM enzymes including two class II lanthipeptide synthetases, two YcaO‐like proteins, a dehydrogenase, a protease/transporter, and a methyltransferase.…”