Lantibiotic transporter requires cooperative functioning of the peptidase domain and the ATP binding domain

Abstract

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics that contain unusual amino acids such as dehydro and lanthionine residues. Nukacin ISK-1 is a class II lantibiotic, whose precursor peptide (NukA) is modified by NukM to form modified NukA. ATP-binding cassette (ABC) transporter NukT is predicted to cleave off the N-terminal leader peptide of modified NukA and secrete the mature peptide. Multiple sequence alignments revealed that NukT has an N-terminal peptidase domain (PEP) and a C-terminal ATP binding domain (ABD). Previously, in vitro reconstitution of NukT has revealed that NukT peptidase activity depends on ATP hydrolysis. Here, we constructed a series of NukT mutants and investigated their transport activity in vivo and peptidase activity in vitro. Most of the mutations of the conserved residues of PEP or ABD resulted in failure of nukacin ISK-1 production and accumulation of modified NukA inside the cells. NukT(N106D) was found to be the only mutant capable of producing nukacin ISK-1. Asn(106) is conserved as Asp in other related ABC transporters. Additionally, an in vitro peptidase assay of NukT mutants demonstrated that PEP is on the cytosolic side and all of the ABD mutants as well as PEP (with the exception of NukT(N106D)) did not have peptidase activity in vitro. Taken together, these observations suggest that the leader peptide is cleaved off inside the cells before peptide secretion; both PEP and ABD are important for NukT peptidase activity, and cooperation between these two domains inside the cells is indispensable for proper functioning of NukT.

FIGURE 1.

Sequence alignment of ABC transporter containing the N-terminal peptidase domain. The amino acid sequence alignments of NukT, LctT, CvaB, LcnC, and ComA were performed using the ClustalW program. The conserved active sites in the peptidase domain are marked with asterisks. The conserved motifs in the ABD (Walker A, Walker B, H-loop) are indicated under the sequence. The mutation points in NukT are shown above the sequence.

FIGURE 2.

Expression levels of NukT and its mutants in L. lactis. The cell-free extract of the recombinant L. lactis strain was prepared as described under “Experimental Procedures,” and 10 μl of the sample was subjected to SDS-PAGE, followed by Western blotting using NukT antiserum. The arrow indicates the position of NukT (80 kDa).

FIGURE 3.

MALDI-TOF/MS spectrum of the cell-free extract of L. lactis NZ9000 (pInukdT). Modified NukA that had accumulated inside the cells was monitored by MALDI-TOF/MS. Each peak corresponds to a protonated, sodium adduct, potassium adduct, and sodium/potassium adduct at m/z 6332.6, 6354.2, 6374.6, and 6395.3, respectively.

FIGURE 4.

Peptidase activity of NukT using ISO or RSO membrane vesicles. Modified His-NukA (3 μm) was incubated with 1 mg/ml NukT membrane. Nukacin ISK-1 produced by cleavage of the leader peptide was detected from its antibacterial activity using the overlay assay method with L. sakei as the indicator strain.

FIGURE 5.

Expression of NukT mutants in S. carnosus and their peptidase activity in vitro. ISO vesicles were prepared from cells of recombinant S. carnosus, and 5 μg of membrane protein was subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining (A). The arrow indicates the position of NukT (80 kDa). Modified His-NukA (3 μm) was incubated with 1 mg/ml NukT membrane. Nukacin ISK-1 produced after the reaction was detected from its antibacterial activity by using the overlay assay method with L. sakei as the indicator strain (B), and the remaining substrates in the reaction mixture were visualized using Western blotting with anti-His tag antibody (C).

FIGURE 6.

Quantitative analysis of nukacin ISK-1 production (black bars) and peptidase activity (white bars) with wild-type NukT and NukT(N106D). Nukacin ISK-1 production was assessed by measuring antibacterial activity (arbitrary units (AU)/ml) against L. sakei as an indicator strain. Peptidase activity was determined by the remaining substrates (modified His-NukA) in the reaction mixture of the in vitro assay, followed by Western blotting using anti-His antibody. The signals of the remaining substrates were quantified by Multi Gauge software. Substrate consumption by wild-type NukT was taken as 100%. This assay was performed in duplicate, and the average values are shown with error bars.