Zn(II) and Cu(II) binding sites of Calcitermin, an antimicrobial peptide found in human airways

Antimicrobial peptides (AMPs) are evolutionarily conserved molecules with antimicrobial and/or immunomodulatory properties. Almost all antimicrobial peptides are cationic and amphipathic. AMPs are widely distributed in many tissues and cells and are potentially present in any species of invertebrate, plant and animal. They act as mediators of innate host defense, being able to target Gram-positive and Gram-negative bacteria as well as fungi and some enveloped viruses. Therefore, they represent a fertile ground for the design of novel therapeutics for the treatment of antibiotic-resistant infections and severe sepsis.[1] Mediators of innate host defense are also found in mucosal secretions of the respiratory tract and include substances, like antimicrobial proteins and (poly)peptides, that can sequester metal nutrients, such as Zn(II), Cu(II), Ni(II), Ca(II), etc.[2-3] Among several uncharacterized molecules that contribute to the overall antimicrobial activity of human nasal fluid, a 15-residue antimicrobial peptide named Calcitermin has been identified.[4] Its sequence (VAIALKAAHYHTHKE) exactly corresponds to the C-terminal domain of Calgranulin C, a calcium-binding pro-inflammatory protein of the S100 family. Several studies have reported that cleavage fragments of parent proteins have potent antimicrobial activity (e.g. buforin I,[5] lactoferricin,[6] KDAMP,[7] vasostatin-1[8]). While Calcitermin does not express, in vitro, any antimicrobial activity in phosphate buffer at pH 7.4, under more acidic conditions (pH 5.4) – which are quite common in inflammatory fluids – it was active against E. coli, P. aeruginosa and C. albicans. Furthermore, it was demonstrated that the presence of micromolar concentrations of Zn(II) enhances its antimicrobial activity against E. coli and L. monocytogenes.[4] Calcitermin contains a putative metal-binding domain with three alternated histidine residues (His9, His11 and His13) and the free terminal amino group. In addition, it has the potential to adopt a helical conformation in membranes. The above results prompted us to deeply investigate the complex-formation equilibria of Calcitermin with Zn(II) and Cu(II), two endogenic and competing metal ions. The unprotected peptide VAIALKAAHYHTHKE has been considered, along with its three mutants, in which each His residue is replaced with one alanine (VAIALKAAAYHTHKE, VAIALKAAHYATHKE, VAIALKAAHYHTAKE). The characterization of the complexes has been achieved by means of mass spectrometry, potentiometry, UV-Vis spectrophotometry, circular dichroism (CD) and nuclear magnetic resonance (NMR). Biological tests were also performed. The comparison among the behaviors of the different analogues helps to shed light on the role played by each His residue in metal coordination geometry and complex stability also in the view of its antimicrobial activity. The preliminary results show that all the investigated peptides are efficient ligands for the considered metal ions and are able to form mono-nuclear complexes where the histidine residues are by far the first metal anchor (Fig.1). References: [1] R. E. W. Hancock, H.-G. Sahl, Nat. Biotechnol. 2006, 24, 1551. [2] K. A. Brogden, Nat. Rev. Microbiol. 2005, 3, 238. [3] T. Ganz, Integr. Comp. Biol. 2003, 43, 300-304. [4] A. M. Cole, Y.-H. Kim, S. Tahk, T. Hong, P. Weis, A. J. Waring, T. Ganz, FEBS Lett. 2001, 504, 5-10. [5] I. Minn, H. S. Kim, S. C. Kim, Biochim. Biophys. Acta - Molecular Basis of Disease 1998, 1407, 31-39. [6] K. Yamauchi, M. Tomita, T. J. Giehl, R. T. Ellison, 3rd, Infect. Immun. 1993, 61, 719-728. [7] C. Tam, J. J. Mun, D. J. Evans, S. M. J. Fleiszig, J. Clin. Investig. 2012, 122, 3665-3677. [8] K. Lugardon, R. Raffner, Y. Goumon, A. Corti, A. Delmas, P. Bulet, D. Aunis, M. H. Metz-Boutigue, J. Biol. Chem. 2000, 275, 10745-10753.