Study of Cu(II) and Zn(II) interaction with the metal binding domain of ZinT protein

Understanding the mechanism of metal trafficking at the host/pathogen interface can help designing innovative antibiotic therapies. In fact, bacteria rely on sophisticated systems (e.g. metallophores) to sequester metals from the host environment during infections. In the attempt to investigate these metal-acquisition processes, we studied the complexation of Cu(II) and Zn(II) – two endogenous and competing metal ions – with some peptide fragments of ZinT, a periplasmic protein found in several bacterial species and mostly involved in Zn(II) recruitment. Its most probable metal-binding site corresponds to a domain containing three histidine residues (positions 167, 176 and 178) and one aspartic acid (position 166, Fig. 1). ZinT also possesses a highly conserved histidine-rich loop (HGHHXH, residues 124-129), whose participation in metals uptake has also been suggested [1,2]. By means of ESI-MS, potentiometry, UV-Vis/CD spectrophotometry and EPR measurements, we studied the formed metal complexes with the protected peptides Ac-124HGHHSH129-Am and Ac-166DHIIAPRKSSHFH178-Am (of ZinT sequence from Escherichia coli), and Ac-124HGHHAH129-Am and Ac-166DHIIAPRKSAHFH178-Am (ZinT-Salmonella enterica). We ultimately compared ZinT with some human-defence mediators, e.g. the antimicrobial peptide Calcitermin [3], to evaluate the metal effectiveness in the expression of the pathogenic/antimicrobial activity by the studied systems. [1] A. Ilari, F. Alaleona, G. Tria, P. Petrarca, A. Battistoni, and C. Zamparelli, Biochim. Biophys. Acta. 1840(1) (2013) 535–544. [2] P. Petrarca, S. Ammendola, P. Pasquali, and A. Battistoni, J. Bacteriol. 192(6) (2010) 1553–1564. [3] D. Bellotti, M. Toniolo, D. Dudek, A. Mikolajczyk, R. Guerrini, A. Matera-Witkiewicz, M. Remelli, and M. Rowinska-Zyrek, Dalton Trans. 48(36), (2109) 13740–13752.