The D-enantiomer of the antimicrobial peptide KLKLLLLLKLK-NH₂ preferentially binds to lipopolysaccharide assemblies...

The all-D-enantiomer of the α-helical antimicrobial peptide KLKLLLLLKLK-NH₂ (DL5), which is based on Sapecin B derived from the flesh fly Sarcophaga peregrina, exhibits superior antimicrobial activity compared to its L-form counterpart (L5). While the enhanced efficacy of D-form peptides is conventionally attributed to their intrinsic resistance to proteolytic degradation, emerging evidence suggests stereoselective interactions with bacterial surface components may independently contribute to their biological activity. To elucidate this mechanism, we investigated the binding properties of these enantiomers to lipopolysaccharide (LPS), the primary structural component of the Gram-negative bacterial outer membrane, utilizing surface plasmon resonance (SPR). DL5 displayed a significantly higher affinity for LPS micelles, exhibiting an approximately 2.52-fold lower apparent dissociation constant than L5. Kinetic analysis further revealed that DL5 dissociated more slowly from LPS, indicating the formation of a highly stable peptide-LPS complex that may prolong interactions at the bacterial surface. Importantly, this enantiomeric divergence in binding affinity was accentuated under physiologically relevant conditions where divalent cations (Mg²⁺ or Ca²⁺) bridge and stabilize the LPS assemblies, regardless of whether they adopt a micellar or a bilayer-like supramolecular morphology. Conversely, disrupting this structural stabilization by chelating endogenous cations with EDTA significantly reduced the affinity difference between the enantiomers. These findings demonstrate that DL5 preferentially recognizes the rigidified, cation-stabilized architecture of LPS aggregates. This specific stereochemical interaction provides a novel structural and mechanistic rationale for the enhanced efficacy of D-form antimicrobial peptides against Gram-negative pathogens.