Lysine-oriented charges trigger the membrane binding and activity of nukacin ISK-1.
The antibacterial activities and membrane binding of nukacin ISK-1 and its fragments and mutants were evaluated to delineate the determinants governing structure-function relationships. The tail region (nukacin(1-7)) and ring region (nukacin(7-27)) were shown to have no antibacterial activity and also had no synergistic effect on each other or even on nukacin ISK-1. Both a fragment with three lysines in the N terminus deleted (nukacin(4-27)) and a mutant with three lysines in the N terminus replaced with alanine (K1-3A nukacin ISK-1) imparted very low activity (32-fold lower than nukacin ISK-1) and also exhibited a similar antagonistic effect on nukacin ISK-1. Addition of two lysine residues at the N terminus (+2K nukacin ISK-1) provided no further increased antibacterial activity. Surface plasmon resonance sensorgrams and kinetic rate constants determined by a BIAcore biosensor revealed that nukacin ISK-1 has remarkably higher binding affinity to anionic model membrane than to zwitterionic model membrane. Similar trends of strong binding responses and kinetics were indicated by the high affinities of nukacin ISK-1 and +2K nukacin ISK-1, but there was no binding of tail region, ring region, nukacin(4-27), and K1-3A nukacin ISK-1 to the anionic model membrane. Our findings therefore suggest that the complete structure of nukacin ISK-1 is necessary for its full activity, in which the N-terminus three lysine residues play a crucial role in electrostatic binding to the target membrane and therefore nukacin ISK-1's ability to exert its potent antibacterial activity.
Asaduzzaman
Sikder M
SM
Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Nagao
Jun-Ichi
J
Aso
Yuji
Y
Nakayama
Jiro
J
Sonomoto
Kenji
K
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D001430:Bacteriocins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D004563:Electrochemistry; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013210:Staphylococcus / Q000235:genetics; D020349:Surface Plasmon Resonance
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D004269:DNA, Bacterial; D008567:Membranes, Artificial; D010446:Peptide Fragments; C421186:nukacin ISK-1; D008239:Lysine