Zn(II) and Cu(II) Binding Ability of ZinT – A Highly Conserved Periplasmic Protein Expressed by Different Bacterial Species

The necessity of new antimicrobial agents is unarguable, since current therapeutic treatments are not always effective due to the development of bacterial drug resistance. Nevertheless, the mechanism of metal trafficking at host/pathogens interface can provide a fertile ground for the design of new effective antibiotic therapies. To prevent infections, humans restrict the access to essential micronutrients by means of an innate immune response termed "nutritional immunity”, on the contrary bacteria rely on sophisticated systems (e.g. metallophores) to overcome the scarce metal bioavailability. In the attempt to shed light on the host nutritional immune response (e.g. deepen the way of action of antimicrobial peptides and other metal-sequestering proteins) and to develop novel highly specific antibiotics, it is crucial to investigate not only the host-mediated defence but also the pathogenic metals acquisition processes [1]. Among several proteins involved in the mechanism of metals recruitment, we recently focused on ZinT, a 216-amino acid protein found in the cytoplasm of several bacterial species, which undergoes translocation to the periplasm in order to express its task of Zn(II) uptake under sever zinclimited conditions, then shuttling the metal to ZnuABC transporter. The most probable metal-binding site of ZinT corresponds to three highly conserved histidine residues (His167, His176 and His178). Additionally, ZinT possesses a highly conserved N-terminal histidine-rich loop (HGHHXH), whose role is unclear, although it has been suggested its participation in Zn(II) uptake [2-4]. The above results prompted us to deeply investigate thermodynamics and coordination chemistry of ZinT complexes with Zn(II) and Cu(II), two endogenic and competing metal ions. For this purpose, we studied the protected peptides Ac-166DHIIAPRKSSHFH178-Am and Ac- 166DHIIAPRKSAHFH178-Am, corresponding to the 166-178 amino acid sequence of ZinT in Escherichia coli and Salmonella enterica and typhimurium, respectively. The N-terminal His-rich sequences of ZinTE. coli (Ac-124HGHHSH129-Am) and ZinT-S.typhimurium (124HGHHAH129-Am) have been also considered, along with the His-rich fragment of YrpE metal-binding protein from Bacillus subtilis (Ac- 57HTHEHSHDHSHAH69-Am), which shows a remarkable similarity with ZinT. 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). Financial support of the National Science Centre (UMO-2017/26/A/ST5/00364) is gratefully acknowledged. 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.