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.

FIG. 1.

Proposed structure of nukacin ISK-1 showing its fragments and mutants used for this study. A-S-A, lanthionine; Abu-S-A, 3-methyllanthionine; Dhb, dehydrobutyrine. Tail region (nukacin_1-7) was synthesized chemically, ring region (nukacin_7-27) was generated by digestion with Pfu N-acetyl deblocking amino peptidase, and nukacin_4-27 fragment was obtained by digestion with endoproteinase Lys-C. K1-3A nukacin ISK-1 and +2K nukacin ISK-1 were generated by genetic engineering.

FIG. 2.

Binding affinity of nukacin ISK-1 to anionic [1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt] and zwitterionic (1,2-dioleyol-sn-glycero-3-phosphocholine) model membranes determined by SPR biosensor. Sensorgrams for 7 μM nukacin ISK-1 bound to each of the anionic (a) and zwitterionic (b) model membranes are indicated.

FIG. 3.

Dose response of nukacin ISK-1 towards the anionic model membrane. Nukacin ISK-1 concentrations were 20 (a), 15 (b), and 5 (c) μM.

FIG. 4.

SPR sensorgrams denoting the binding of +2K nukacin ISK-1 (a), nukacin ISK-1 (b), tail region (c), ring region (d), nukacin_4-27 (e), and K1-3A nukacin ISK-1 (f) to the anionic model membrane. The concentration of nukacin ISK-1 and its derivatives was 7 μM.