Molecular mechanism of target recognition by subtilin, a class I lanthionine antibiotic

Abstract

The increasing resistance of human pathogens to conventional antibiotics presents a growing threat to the chemotherapeutic management of infectious diseases. The lanthionine antibiotics, still unused as therapeutic agents, have recently attracted significant scientific interest as models for targeting and management of bacterial infections. We investigated the action of one member of this class, subtilin, which permeabilizes lipid membranes in a lipid II-dependent manner and binds bactoprenyl pyrophosphate, akin to nisin. The role the C and N termini play in target recognition was investigated in vivo and in vitro by using the natural N-terminally succinylated subtilin as well as enzymatically truncated subtilin variants. Fluorescence dequenching experiments show that subtilin induces leakage in membranes in a lipid II-dependent manner and that N-succinylated subtilin is roughly 75-fold less active. Solid-state nuclear magnetic resonance was used to show that subtilin forms complexes with membrane isoprenyl pyrophosphates. Activity assays in vivo show that the N terminus of subtilin plays a critical role in its activity. Succinylation of the N terminus resulted in a 20-fold decrease in its activity, whereas deletion of N-terminal Trp abolished activity altogether.

FIG. 1.

Structures of subtilin (a) and N-succinylated subtilin (b). Arrows indicate points of cleavage that generate fragments.

FIG. 2.

Mass spectra of subtilin, N-succinylated subtilin, and subtilin fragments. (A) Subtilin, m/z 3,321 Da; (B) N-succinylated subtilin, m/z 3,421 Da; (C) subtilin fragment 1-29, m/z 3,010 Da; (D) subtilin fragment 2-29, m/z 2,824 Da. The observed molecular ion at m/z 2,466 Da is due to an internal reference, human adrenocorticotropic hormone fragment 18-39.

FIG. 3.

Activity of subtilin and subtilin fragments (⧫, subtilin; ▴, subtilin 1-29; ▪, subtilin 2-29) against DOPC-DOPG vesicles with 0.1 mol% lipid II. Peptides induce a leakage of CF from the vesicles.

FIG. 4.

Activity of N-succinylated subtilin against DOPC-DOPG vesicles with 0.1 mol% lipid II. Peptides induce a leakage of CF from the vesicles.

FIG. 5.

Phosphorus-31 NMR spectra of lipid bilayers containing geranylgeranyl pyrophosphate. (A) Spectrum acquired using single-pulse excitation, followed by proton decoupling under 4.7 kHz MAS; (B) spectrum from membranes treated with subtilin and acquired by using tangent ramp cross-polarization, followed by proton decoupling under 5 kHz MAS. A numerical fit to the pyrophosphate spectrum is shown below the spectrum in panel B to guide the eye.

FIG. 6.

Proposed mechanism of pyrophosphate-mediated target engagement by the lanthionine antibiotic subtilin. Membrane breach occurs in a lipid II-mediated fashion and cell wall synthesis is impeded through engagement of pyrophosphate-containing intermediates into complexes.