Zn(II) and Cu(II) interaction with the periplasmic protein ZinT: finding a correlation between coordination chemistry and pathogenic activity

The mechanism of metal trafficking at the host/pathogen interface represent a rational chance to overcome the current drug-resistance crisis and ultimately design innovative antimicrobial agents. Metals participate in infectious events and are fundamental for the subsistence of pathogenic microorganisms. On the other hand, also humans (i.e. the host organisms) need metal ions to ensure the correct performance of their biological functions: a sort of ‘tug-of-war’ is therefore established between the host and the pathogen for the metals acquisition. As a consequence, in the attempt to develop novel highly specific metal-based antibiotics, it is crucial to investigate not only the host-mediated defence but also the metal-acquisition strategies put in place by pathogens [1]. Among several metal-sequestering proteins involved in these processes, we recently focused on ZinT, a 216-amino acid protein mostly expressed by Gram-negative bacteria. ZinT undergoes translocation to the periplasm in order to bind Zn(II) under strict zinc-limited conditions and shuttle the metal to ZnuABC transporter [2]. The most probable metal-binding site of ZinT corresponds to a domain containing three highly conserved histidine residues (position 167, 176 and 178) and one aspartic acid (position 166). Additionally, ZinT possesses a highly conserved N-terminal histidine-rich loop (HGHHXH, residues 124-129), whose role is unclear, although its participation in Zn(II) uptake has been suggested [3,4]. The above results prompted us to deeply investigate complexation of Cu(II) and Zn(II) – two endogenous and competing metal ions – with these domains of ZinT. We studied the protected peptides Ac-124HGHHSH129-Am and Ac-166DHIIAPRKSSHFH178-Am belonging to the amino acid sequence of the ZinT protein from Escherichia coli, and Ac-124HGHHAH129-Am and Ac-166DHIIAPRKSAHFH178-Am from the ZinT expressed by Salmonella enterica. The characterization of the complexes has been achieved by means of mass spectrometry, potentiometry, UV-Vis spectrophotometry, circular dichroism (CD) and electronic paramagnetic resonance (EPR). The preliminary results show that all the investigated peptides are efficient ligands for the considered metal ions and are able to form stable mono-nuclear complexes where the histidine residues and, if present, the aspartic acid participate in the metal coordination (Figure 1). We ultimately compared ZinT with some human-defence mediators, e.g. the antimicrobial peptide Calcitermin [6], to evaluate the metal effectiveness in the expression of the pathogenic/antimicrobial activity by the studied systems. REFERENCES [1] P. Chandrangsu, C. Rensing, J. D. Helmann, Nat. Rev. Microbiol. 15(6) (2017), 338–350. [2] A. Ilari, F. Alaleona, G. Tria, P. Petrarca, A. Battistoni, C. Zamparelli, Biochim. Biophys. Acta. 1840(1) (2013) 535–544. [3] P. Petrarca, S. Ammendola, P. Pasquali, A. Battistoni, J. Bacteriol. 192(6), (2010) 1553–1564. [4] J. Chen, L. Wang, F. Shang, Y. Dong, N.-C. Ha, K.H. Nam, C. Quan, Y. Xu, Biochem. Biophys. Res. Commun. 500(2), (2018) 139–144. [5] The PyMOL Molecular Graphics System, Version 1.8, Schrödinger, LLCD. [6] 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.