A Single L17E Mutation Switches the Membrane Disruption Mechanism of the Spider Venom Peptide M-lycotoxin

Membrane-active peptides (MAPs) disrupt lipid bilayers through mechanisms that depend sensitively on amino acid sequence, yet the molecular determinants governing membrane pore formation remain incompletely understood. The spider-venom peptide M-lycotoxin exhibits strong membrane-disruptive activity, whereas a single substitution of Leu17 with glutamate (L17E) markedly reduces cytotoxicity. Here, we employed microsecond-scale all-atom molecular dynamics simulations to investigate how this mutation alters the membrane disruption pathway. The wild-type (WT) peptide preserves strong amphiphilicity and stable α-helicity, enabling deep insertion into the membrane. In multiple independent simulations, WT peptides spontaneously nucleated membrane defects that evolved into toroidal pores stabilized by inward bending of lipid headgroups and cooperative insertion of a small number of peptides. These pores contain continuous water columns and permit transient ion permeation. In contrast, the L17E mutation disrupts amphiphilicity and reduces helical stability, weakening peptide-membrane coupling. As a result, spontaneous pore formation is not observed. Under an applied electric field used to accelerate rare membrane penetration events in simulations, the mutant adopts only a largely nonconductive transmembrane configuration that does not develop into cooperative pore assemblies. These results indicate that the L17E mutation shifts the membrane disruption pathway from cooperative toroidal pore formation to a nonconductive transmembrane state, providing a molecular explanation for the reduced cytotoxicity of the mutant.