Lysine-Oriented Charges Trigger the Membrane Binding and Activity of Nukacin ISK-1

Abstract

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.

Fundamental functionality: recent developments in understanding the structure–activity relationships of lantibiotic peptides

“…The N terminus of this peptide contains three cationic lysine residues (giving nukacin ISK-1 an overall plus-three charge) that were postulated to be vital for interaction with the cell membrane and thus the antibacterial activity. 30 Mutants were produced that abolished the overall charge of nukacin ISK-1 both by deletion of the first three lysine residues and by substitution of all the three with alanine residues (see Figure 4). These changes resulted in a large decrease in activity compared with wild-type nukacin ISK-1.…”

“…Indeed in their earlier study, the authors discussed an analog with two further lysine residues added to nukacin ISK-1 that showed no increase in antibacterial activity, suggesting that the magnitude of charge may be less important so long as an overall positive charge exists. 30 The first analogs of the AII group to achieve enhanced activity when compared with the wild-type peptide were produced for nukacin ISK-1. Asp13Glu and Val22Ile were both found to display specific activities more potent than nukacin ISK-1 itself.…”

Lantibiotic Resistance

“…In the case of many cationic lantibiotics, or lantibiotics with cationic domains, there is an initial attraction to the anionic cell membrane of target microbes (6)(7)(8). Notably, changes in lantibiotic charge as a result of genetic engineering alter the efficacy of such lantibiotics, presumably due to a reduced attraction/interaction with the cell membrane (8,9).…”

Lantibiotic Transporter Requires Cooperative Functioning of the Peptidase Domain and the ATP Binding Domain

“…A 10-ml volume of the culture supernatant was loaded onto a Sep-Pak C 18 cartridge (Waters) for partial purification, and the eluate was applied to liquid chromatography/mass spectrometry (LC/ MS) coupled to an AccuTOF T100LC mass spectrometer (JEOL, Tokyo, Japan). The relative productivity of nukacin ISK-1 was assayed by the spot-on-lawn method (25). Lactobacilli Agar AOAC (Becton Dickinson) was overlaid on MRS agar medium with L. sakei subsp.…”