Molecular mechanism of target recognition by subtilin, a class I lanthionine antibiotic.
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.
Parisot
Judicaël
J
School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, United Kingdom.
Carey
Sarah
S
Breukink
Eefjan
E
Chan
Weng C
WC
Narbad
Arjan
A
Bonev
Boyan
B
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001430:Bacteriocins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018699:Coated Vesicles / Q000502:physiology; D011756:Diphosphates / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D013294:Lactococcus lactis / Q000187:drug effects*; D009682:Magnetic Resonance Spectroscopy; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D019802:Succinic Acid / Q000378:metabolism; D013440:Sulfides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014364:Tryptophan / Q000378:metabolism; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D011756:Diphosphates; D005452:Fluoresceins; D010455:Peptides; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D013440:Sulfides; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; C024098:6-carboxyfluorescein; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D014364:Tryptophan; D019802:Succinic Acid; C017251:1,2-oleoylphosphatidylcholine; C002836:subtilin; C001520:lanthionine; D000409:Alanine