Understanding the thermodynamics and coordination chemistry of metal-binding proteins: the common thread to elucidate metal acquisition processes at host/pathogen interface

Several studies have shown that the disruption of metal homeostasis in bacterial and fungal cells can be a powerful tool to design new antimicrobial drugs with a high rate of selectivity and specificity. To prevent infections, in fact, the human organism reduces the bioavailability of essential micronutrients by means of an innate immune response termed “nutritional immunity”; on the contrary, pathogens rely on specialized metal-binding proteins and molecular systems which capture the metal ions from the competitive host environment forming stable complexes [1]. Understanding the properties, structure and action mechanisms of the involved metal chelators is the very first step to elucidate the dynamics behind the metal transfer mechanisms and to rationally design novel metal-based antibiotic therapeutics [2]. An outstanding example is given by the thermodynamic and spectroscopic characterization of the zinc and copper binding sites of the periplasmic protein ZinT, expressed by Escherichia coli and Salmonella enterica [3, 4]. The chosen unstructured fragments, which serve as models to simulate the coordination and transport of metal ions in ZinT protein, correspond to the 24–29 and 166–178 amino acid sequences and are protected at their amino- and carboxyl-termini: Ac-24HGHHSH29-Am and Ac-166DHIIAPRKSSHFH178-Am (E. coli), Ac-24HGHHAH29-Am and Ac- 166DHIIAPRKSAHFH178-Am (S. enterica). A deep investigation on the thermodynamics and coordination chemistry of the formed Zn(II) and Cu(II) complexes was performed through different experimental techniques. The protonation and complex-formation equilibria were studied by means of potentiometric acid-base titrations. ESI mass spectra of the solutions under examination allowed to confirm the stoichiometries of the formed species and, through UV-Vis, CD and EPR spectroscopies at variable pH values, the metal coordination spheres and the geometry of the complexes were explored. Finally, the obtained results allowed a comparison with other biologically relevant metal-binding systems, such as the antimicrobial peptide calcitermin (VAIALKAAHYHTHKE) which can, in principle, participate in human nutritional immunity, competing with ZinT for the metal ion acquisition. Financial support of the National Science Centre (UMO-2020/37/N/ST4/03165) is gratefully acknowledged. This paper is based upon work from COST Action CA18202, NECTAR – Network for Equilibria and Chemical Thermodynamics Advanced Research, supported by COST (European Cooperation in Science and Technology). References [1] D. A. Capdevila, K. A. Edmonds, D. P. Giedroc, Essays Biochem., 2017, 61(2), 177-200. [2] S. R. Hennigar, J. P. McClung, Am. J. Lifestyle Med., 2016, 10(3), 170-173. [3] A. Battistoni, A. Ammendola, E. Chiancone, A. Ilari, Future Med. Chem., 2017, 9(9), 899-910. [4] D. Bellotti, M. Rowińska-Żyrek, M. Remelli, Dalton Trans., 2020, 49(27), 9393-9403.