ZinT is a periplasmic protein found in Gram-negative bacteria. It is involved in cellular metal trafficking and may function as zinc-chaperone for the ZnuABC transporter. There is a general consensus that the three highly conserved histidine residues (His167, His176 and His178) facing the centre of ZinT calycin like-domain and the amino-terminal fragment between residues 24 and 29 (-HXHHXH-) should be effective zinc binding sites [1, 2]. The Zn2+-ZinT complex from S. enterica can interact with ZnuA forming a ternary complex where both proteins expose their binding pocket to the Zn2+ ion and where the His-rich loop of ZnuA functions as a hypothetical metal transfer intermediary between the two proteins [3, 4]. The main aim of this work is therefore to provide insight into the correlation between the metal-binding ability of ZinT and its biological role. The chosen unstructured fragments, which serve as models to simulate the coordination and transport of metal ions in ZinT protein (see Figure 1) from Escherichia coli and Salmonella enterica, correspond to the 24–29 and 166–178 amino acid sequences and are protected at the amino- and carboxyl-termini: Ac-24HGHHSH29-Am and Ac-166DHIIAPRKSSHFH178-Am (E. coli), Ac-24HGHHAH29-Am and Ac-166DHIIAPRKSAHFH178-Am (S. enterica). Interestingly, both the metal-binding sites of ZinT from S. enterica undergo a Ser-to-Ala substitution (position 28 and 175). A deep investigation of the complex-formation equilibria and coordination chemistry of the formed species has been performed through different experimental techniques, including potentiometry, mass spectrometry and various spectroscopies. The obtained results highlight novel insight into the mechanism of ZinT-mediated metal acquisition and allow a comparison with other biologically relevant metal-binding systems, such as the antimicrobial peptide calcitermin 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-2017/26/A/ST5/00364 and 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] J. Chen, L. Wang, F. Shang, Y. Dong, N.-C. Ha, K. H. Nam, C. Quan, Y. Xu, Biochem. Biophys. Res. Commun. 2018, 500(2), 139-144. [2] H. G. Colaço, P. E. Santo, P. M. Matias, T. M. Bandeiras, J. B. Vicente, Metallomics 2016, 8(3), 327-336. [3] A. Ilari, F. Alaleona, G. Tria, P. Petrarca, A. Battistoni, C. Zamparelli, D. Verzili, M. Falconi, E. Chiancone, Biochim. Biophys. Acta 2014, 1840(1), 535-544. [4] D. Bellotti, M. Rowińska-Żyrek, M. Remelli, Dalton Trans. 2020, 49(27), 9393-9403.

Understanding the mechanism of ZinT-mediated metal acquisition: a thermodynamic study

Denise BELLOTTI
Primo
;
Maurizio REMELLI
Ultimo
2021

Abstract

ZinT is a periplasmic protein found in Gram-negative bacteria. It is involved in cellular metal trafficking and may function as zinc-chaperone for the ZnuABC transporter. There is a general consensus that the three highly conserved histidine residues (His167, His176 and His178) facing the centre of ZinT calycin like-domain and the amino-terminal fragment between residues 24 and 29 (-HXHHXH-) should be effective zinc binding sites [1, 2]. The Zn2+-ZinT complex from S. enterica can interact with ZnuA forming a ternary complex where both proteins expose their binding pocket to the Zn2+ ion and where the His-rich loop of ZnuA functions as a hypothetical metal transfer intermediary between the two proteins [3, 4]. The main aim of this work is therefore to provide insight into the correlation between the metal-binding ability of ZinT and its biological role. The chosen unstructured fragments, which serve as models to simulate the coordination and transport of metal ions in ZinT protein (see Figure 1) from Escherichia coli and Salmonella enterica, correspond to the 24–29 and 166–178 amino acid sequences and are protected at the amino- and carboxyl-termini: Ac-24HGHHSH29-Am and Ac-166DHIIAPRKSSHFH178-Am (E. coli), Ac-24HGHHAH29-Am and Ac-166DHIIAPRKSAHFH178-Am (S. enterica). Interestingly, both the metal-binding sites of ZinT from S. enterica undergo a Ser-to-Ala substitution (position 28 and 175). A deep investigation of the complex-formation equilibria and coordination chemistry of the formed species has been performed through different experimental techniques, including potentiometry, mass spectrometry and various spectroscopies. The obtained results highlight novel insight into the mechanism of ZinT-mediated metal acquisition and allow a comparison with other biologically relevant metal-binding systems, such as the antimicrobial peptide calcitermin 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-2017/26/A/ST5/00364 and 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] J. Chen, L. Wang, F. Shang, Y. Dong, N.-C. Ha, K. H. Nam, C. Quan, Y. Xu, Biochem. Biophys. Res. Commun. 2018, 500(2), 139-144. [2] H. G. Colaço, P. E. Santo, P. M. Matias, T. M. Bandeiras, J. B. Vicente, Metallomics 2016, 8(3), 327-336. [3] A. Ilari, F. Alaleona, G. Tria, P. Petrarca, A. Battistoni, C. Zamparelli, D. Verzili, M. Falconi, E. Chiancone, Biochim. Biophys. Acta 2014, 1840(1), 535-544. [4] D. Bellotti, M. Rowińska-Żyrek, M. Remelli, Dalton Trans. 2020, 49(27), 9393-9403.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2478996
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