Molecular action of NZ2114, a superior plectasin derivative

NZ2114 is a triple-mutant of the natural peptide antibiotic plectasin that targets the peptidoglycan precursor molecule Lipid II using a supramolecular action, involving assembly of plectasin-Lipid II complexes in a large oligomeric structure, that is enhanced by calcium ions. Due to its superior potency against Staphylococcus aureus strains, NZ2114 was the candidate that was advanced to clinical trials, and it has become the standard-template for the development of improved plectasin derivatives. However, the molecular underpinning for NZ2114's improved potency remains opaque, with biochemical data pointing to chemical modification of the Lipid II target in Staphylococci that would impair the target binding capacity of plectasin but not the one of NZ2114. Here, using an integrative structural biology approach based on solid-state NMR, high-speed atomic force microscopy, and affinity assays, we demonstrate that both NZ2114 and plectasin bind effectively to Staphylococcal Lipid II variants, which means that NZ2114's greater potency against S. aureus does not result from a difference in target binding. Instead, we show that the three residue substitutions in NZ2114 change its N-terminal fold, markedly increasing its sensitivity to calcium ions, which results in a different supramolecular action on the membrane surface. Altogether, our study provides new insights for the design of superior drug candidates.