Pore formation by nisin involves translocation of its C-terminal part across the membrane.
Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.
van Kraaij
C
C
Microbial Ingredients Section, NIZO food research, Ede, The Netherlands. vankraay@nizo.nl
Breukink
E
E
Noordermeer
M A
MA
Demel
R A
RA
Siezen
R J
RJ
Kuipers
O P
OP
de Kruijff
B
B
D000595:Amino Acid Sequence; D000838:Anions; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009561:Nisin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D000838:Anions; D000900:Anti-Bacterial Agents; D008563:Membrane Lipids; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D009561:Nisin; D006639:Histidine; C071364:nisin Z