Activity Determinants in Linear Spider Venom Peptide Fragments Against MRSA

Abstract Objective: Using neural networks, we previously identified linear fragments of spider venom peptides active against methicillin-resistant Staphylococcus aureus (MRSA). Among them, peptide IX (IWLSLMKFAGKHL-NH2) with a C-terminal amide displayed high antibacterial potency, whereas its non-amidated analogue, peptide X, was inactive. Peptide IX incorporates into zwitterionic multilamellar liposomes of dioleoylphosphatidylcholine (DOPC) without disrupting them, and also into anionic liposomes of dioleoylphosphatidylglycerol (DOPG) mimicking the MRSA cell membrane, leading to bilayer disruption. This study aims to elucidate the reasons for peptide X inactivation. Methods: According to ¹H NMR spectroscopy, peptide X in water is governed by the ionization of the His12 residue. ³¹P NMR data show that peptide X interacts with DOPC and DOPG membranes. Monte Carlo simulations revealed conformational differences between peptides IX and X upon interaction with an implicit membrane model. Results and Discussion: At pH 7.0 (net charge +2), peptide X aggregates, whereas at pH 5.0 (charge +3), it is monomeric. In contrast, peptide IX (+3 at pH 7.0 and +4 at pH 5.0) does not aggregate in this pH range. ³¹P NMR data show that peptide X has a weaker effect on bilayer packing than peptide IX and does not disrupt the bilayer in either case, consistent with its lack of anti-MRSA activity at pH 5–7. Monte Carlo simulations revealed conformational differences between peptides IX and X. We propose that the inactivation of peptide X arises from both reduced affinity and suboptimal binding to anionic bilayers, driven by aggregation in solution and weaker electrostatic peptide–membrane interactions. Conclusions: Thus, C-terminal amidation is a key modification required for anti-MRSA activity of short linear antimicrobial peptides.