Sympathy for the serine: the effect of Ala-to-Serine substitution on the metal binding affinity of calcitermin peptide

Nowadays the phenomenon of antimicrobial resistance (AMR) is one of the most urgent threats to public health, causing serious problems in the prevention and successful treatment of many diseases. Misusing and overusing antibacterial agents are considered the major reasons behind the drug-resistance emergence, together with spontaneous evolution and mutation of pathogenic microorganisms. In order to overcome the global AMR crisis, the use of antimicrobial peptides (AMPs) represent a promising strategy for the design of new drugs. Their activity can be expressed in different ways, including the interaction with cell membranes and through the innate immune response termed “nutritional immunity”. Thanks to this mechanism, AMPs sequestrate essential metal micronutrients such as Zn(II), Cu(II), Mn(II), Fe(II) or Ca(II), which are fundamental for pathogen subsistence. Among several AMPs, we are interested in calcitermin,1 a human, a 15 amino-acid antimicrobial peptide: VAIALKAAHYHTHKE. Calcitermin present an effective metal-binding domain with three alternated histidine residues (His9, His11 and His13) and the free terminal amino and carboxyl groups. It exhibits an increased microbicide activity when Zn(II) or Cu(II) ions are present in the culture medium. In order to improve the metal binding ability and the biologic activity of calcitermin, we synthesized and studied the analogues VAIALKSAHYTHKE(A7S),VAIALKASHYHTKE(A8S) and VAIALKSSHYHTHKE(A7S/A8S), in which the alanine residues in position 7 and/or 8 have been replaced with a serine. In fact, several studies carried out on metal chelating peptides have previously shown that the presence of one or more serine residues in the proximity of coordinated histidines stabilized the Cu2+ complexes when the metal ion begins to interact with the amides of the peptide chain.2 This behaviour would improve the metal chelating capacity of our Ala-to-Ser analogues towards copper at physiological and alkaline pH. We studied also the complexes with zinc ion, which is the most important endogenous metal ion capable to interaction with calcitermin. The characterization of the complexes has been achieved by means of potentiometry and UV-Vis spectrophotometry. The results show that all the investigated peptides are efficient ligands for the considered metal ions and are able to form stable mono-nuclear complexes. Financial support of the Polish National Science Centre (UMO-2020/37/N/ST4/03165), of the University of Ferrara (FAR 2021) and of the COST Action CA18202-Nectar is gratefully acknowledged.