The importance of being positive! Ala-to-Arg substitution in the antimicrobial peptide calcitermin

Antimicrobial resistance (AMR) is one of the most important health challenges of the last decades. The number of pathogenic microorganisms capable to adapt and resist against current therapeutic treatments has significantly increased, thus requiring innovative therapies. Among several novel approaches, antimicrobial peptide (AMPs) represents a promising category of new drugs. Their activity can be expressed through different mechanisms, including the interaction with pathogen membranes and “nutritional immunity”, i.e. the sequestration of metal ions, which are fundamental for pathogen subsistence. Calcitermin is a 15-mer peptide (VAIALKAAHYHTHKE) corresponding to the C-terminal domain of calgranulin C, a pro-inflammatory protein of the S100 family. It contains an effective metalbinding domain with three alternated histidine residues (His9, His11 and His13) and the free terminal amino and carboxyl groups [1]. Moreover, it has the potential to adopt helical conformation in membranes, and Zn2+ and Cu2+ ions can enhance its antimicrobial activity against Candida albicans [2] and common bacteria like Staphylococcus aureus and Enterococcus faecalis [3]. In order to improve the biological activity of calcitermin, we synthesized and studied the analogues VAIALKRAHYHTHKE (A7R) and VAIALKARHYHTHKE (A8R), in which the alanine residues in position 7 or 8 have been replaced with an arginine. In fact, a higher number of arginine residues increases the positive charge of the peptide, this favoring the attraction towards the anionic bacterial membrane [4]. In turn, this helps membrane permeabilization (see Fig. 1), resulting in a higher antibacterial activity. The complex-formation equilibria between the above calcitermin derivatives and Cu2+ and Zn2+, two important endogenous metal ions, have been deeply investigated, by potentiometry, UV-Vis spectrophotometry, circular dichroism, electron paramagnetic resonance and mass spectrometry. The results show that the Ala-to-Arg substitution do not significantly affect the stoichiometry and the structure of formed complexes, although the studied peptides show an affinity for the Zn2+ ion higher than that of wild-type calcitermin. Financial support of the Polish National Science Centre (UMO-2020/37/N/ST4/03165), of the University of Ferrara (FIRD 2022 and FAR 2023) and of the COST Action CA18202-Nectar is gratefully acknowledged.