Hydrogen bonding and membrane anchoring of the antimicrobial peptide NP-3a investigated through molecular dynamics

Abstract

Antimicrobial peptides (AMPs) are emerging as critical alternatives to antibiotics in the fight against multidrug resistance. NP-3a, a rabbit defensin, combines structural stability with broad-spectrum activity, yet its molecular mechanism of membrane interaction remains unclear. Here, we employed atomistic molecular dynamics simulations to investigate NP-3a in vacuum, aqueous solution, and at a DOPC lipid bilayer interface. In solution, NP3a shifted from a compact β-sheet stabilized by ~23 intramolecular HBs to a dynamic state engaging extensively with water (~122 HBs, lifetime ~9.6 ps). At the membrane interface, NP-3a achieved stable anchoring with ~39% insertion, mediated by ~12 long-lived hydrogen bonds (~2.9 ns lifetime) with DOPC headgroups and a binding free energy of 24.3 kJ/mol. Residue-level analysis revealed Arg-7 to Arg-9 as dominant contributors through electrostatic anchoring to phosphate groups, reinforced by serine- and cysteine-mediated contacts. Notably, NP-3a remained localized at the membrane surface without penetrating the hydrophobic core, supporting a selective surface-associated mechanism of action. These findings provide atomistic insights into NP-3a’s interaction with eukaryotic-like membranes and highlight molecular determinants relevant for the rational design of next-generation AMPs.