Does the Ala-to-Ser substitution affect the metal-binding and biological properties of the antimicrobial peptide calcitermin?

The phenomenon of antimicrobial resistance (AMR) occurs when microorganisms become able to adapt and grow in the presence of previously effective medications. Misusing and overusing antibacterial agents are considered the major reasons behind the resistance emergence, together with spontaneous evolution and mutation of pathogens. In order to tackle this global emergence, the use of antimicrobial peptides (AMPs) represent a promising strategy for the design of new drugs. AMPs can act through different mechanisms, including the interaction with cell membranes and the innate immune response termed “nutritional immunity”. Thanks to this mechanisms, 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 [1,2], a human 15 amino-acid antimicrobial peptide: VAIALKAAHYHTHKE. Calcitermin contains an effective metal binding domain with three alternated histidine residues (His 9, His 11 and His 13) 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 affinity and the biologic activity of calcitermin, we synthesized and studied the analogues VAIALKSAHYHTHKE (A7S), VAIALKASHYHTHKE (A8S) and VAIALKSSHYHTHKE (A7S/A8S), in which the alanines in position 7 and/or 8 have been replaced by serines. 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 stabilizes the Cu2+ complexes when the metal ion begins to interact with the amides of the peptide chain [3]. We also studied the complexes with zinc ion, which is the most important endogenous metal ion capable to interact with calcitermin. The characterization of the complexes has been achieved by means of mass spectrometry, potentiometry, UV-Vis spectrophotometry, circular dichroism, electron paramagnetic resonance. The proteolytic stability of these three peptides has been tested in human plasma by means of HPLC, evaluating the degradation after different times of incubation at 37°C.