Nutritional immunity is the “process by which a host organism sequesters trace minerals in an effort to limit pathogenicity during infection” [1]. All living organisms need trace minerals to synthesize metalloproteins and metalloenzymes which are essential for biological processes, such as oxygen transport, protection from oxidative stress or, as in the case of Helicobacter pylori, the synthesis of ammonia for buffering the pH of its living environment. Pathogens must acquire these trace minerals from their host which, on the other hand, defends himself by putting in place effective competition actions. Studying the mechanisms of transport and supply of transition metals by bacteria can provide weapons to fight infections, helping the processes of nutritional immunity and overcoming the problems caused by the increasingly widespread resistance to classic antibiotic therapies. H. pylori is a human gastrointestinal pathogen that can be responsible for disorders like gastritis, duodenal ulcers and even gastric cancer. For its survival in the human stomach, H. pylori needs two enzymes, urease and Ni-Fe hydrogenase, both containing Ni(II) ions as cofactors. On the other hand, an excess of nickel is potentially toxic. Hpn is a His-rich protein expressed by H. pylori that plays an important role in nickel homeostasis and protects the bacterium from too high concentrations of metal ions. This polypeptide of 59 amino acid residues is abundant in the cytoplasm of H. pylori and represents approximately the 2% of all the proteins synthesized by this bacterium. Recent studies [2] have shown that the N-terminal part of Hpn (MAHHEEQHG-) binds the Ni(II) and Cu(II) ions in the same way as human albumin, since it contains an ATCUN (Amino Terminal Cu and Ni binding)-type sequence, which allows the formation of complexes involving a set of four nitrogens as donor atoms: {NH2, 2N-, Nim}. Starting from these results, the aim of the present work was to investigate the role of the individual histidines in the coordination of Cu(II) and Ni(II) by the N-terminal domain of the Hpn protein. Some model peptides were considered, corresponding to the N-terminal "wild-type" sequence and its analogues in which one or more histidines were replaced by alanine: MAHHEEQHG-NH2 (WT, wild-type Hpn), MAAHEEQHG-NH2 (H3A), MAHAEEQHG-NH2 (H4A), MAHHEEQAG-NH2 (H8A), MAHAEEQAG-NH2 (H4A/H8A). The protonation and complex-formation equilibria were studied by means of potentiometric acid-base titrations and the analysis of the experimental data was obtained through the use of specific calculation programs [3-5]. From the mass spectra of the solutions under examination it was possible to obtain confirmations of the stoichiometries of the formed complexes and through UV-Vis, CD and EPR spectroscopies information was acquired on the coordination geometries and on the set of donor atoms. All the peptide sequences resulted strong ligands for Cu(II) and Ni(II), including the H3A peptide, although it lacks histidine in position 3, characteristic of the ATCUN-type site. For all ligands, the presence of two or more histidine residues in the sequence increases the stability of the metal complexes and also allows the formation of binuclear species. The latter have been studied by carrying out experiments in the presence of excess of metal. The results of this investigation suggest that, in the case of the wild-type peptide (MAHHEEQHG-NH2), at a Cu(II)/ligand ratio of about 2:1 and in alkaline solution, a copper ion is bound to the N-terminal ATCUN site and the second one is instead anchored to the histidine residue in position 8 in the C-terminal domain (see Figure 1.). If Cu(II) and Ni(II) are simultaneously present in solution, the formation of ternary complexes is observed. [1] S.R. Hennigar, J.P. McClung, Am. J. Lifestyle Med., 2016, 10(3), 170–173. [2] D. Witkowska, S. Bielinska, W. Kamysz, H. Kozlowski, J. Bioinorg. Chem., 2011, 105(2), 208-214. [3] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Coord. Chem. Rev., 1999, 184, 311-318. [4] P. Gans, B. O'Sullivan, Talanta, 2000, 51, 33-37. [5] P. Gans, A. Sabatini, A. Vacca, Talanta, 1996, 43, 1739-1753.
Helicobacter pylori and metal ions: solution equilibria studies can help to find new therapies
Maurizio REMELLI
;Denise BELLOTTI;Angelica SINIGAGLIA;Remo GUERRINI
2020
Abstract
Nutritional immunity is the “process by which a host organism sequesters trace minerals in an effort to limit pathogenicity during infection” [1]. All living organisms need trace minerals to synthesize metalloproteins and metalloenzymes which are essential for biological processes, such as oxygen transport, protection from oxidative stress or, as in the case of Helicobacter pylori, the synthesis of ammonia for buffering the pH of its living environment. Pathogens must acquire these trace minerals from their host which, on the other hand, defends himself by putting in place effective competition actions. Studying the mechanisms of transport and supply of transition metals by bacteria can provide weapons to fight infections, helping the processes of nutritional immunity and overcoming the problems caused by the increasingly widespread resistance to classic antibiotic therapies. H. pylori is a human gastrointestinal pathogen that can be responsible for disorders like gastritis, duodenal ulcers and even gastric cancer. For its survival in the human stomach, H. pylori needs two enzymes, urease and Ni-Fe hydrogenase, both containing Ni(II) ions as cofactors. On the other hand, an excess of nickel is potentially toxic. Hpn is a His-rich protein expressed by H. pylori that plays an important role in nickel homeostasis and protects the bacterium from too high concentrations of metal ions. This polypeptide of 59 amino acid residues is abundant in the cytoplasm of H. pylori and represents approximately the 2% of all the proteins synthesized by this bacterium. Recent studies [2] have shown that the N-terminal part of Hpn (MAHHEEQHG-) binds the Ni(II) and Cu(II) ions in the same way as human albumin, since it contains an ATCUN (Amino Terminal Cu and Ni binding)-type sequence, which allows the formation of complexes involving a set of four nitrogens as donor atoms: {NH2, 2N-, Nim}. Starting from these results, the aim of the present work was to investigate the role of the individual histidines in the coordination of Cu(II) and Ni(II) by the N-terminal domain of the Hpn protein. Some model peptides were considered, corresponding to the N-terminal "wild-type" sequence and its analogues in which one or more histidines were replaced by alanine: MAHHEEQHG-NH2 (WT, wild-type Hpn), MAAHEEQHG-NH2 (H3A), MAHAEEQHG-NH2 (H4A), MAHHEEQAG-NH2 (H8A), MAHAEEQAG-NH2 (H4A/H8A). The protonation and complex-formation equilibria were studied by means of potentiometric acid-base titrations and the analysis of the experimental data was obtained through the use of specific calculation programs [3-5]. From the mass spectra of the solutions under examination it was possible to obtain confirmations of the stoichiometries of the formed complexes and through UV-Vis, CD and EPR spectroscopies information was acquired on the coordination geometries and on the set of donor atoms. All the peptide sequences resulted strong ligands for Cu(II) and Ni(II), including the H3A peptide, although it lacks histidine in position 3, characteristic of the ATCUN-type site. For all ligands, the presence of two or more histidine residues in the sequence increases the stability of the metal complexes and also allows the formation of binuclear species. The latter have been studied by carrying out experiments in the presence of excess of metal. The results of this investigation suggest that, in the case of the wild-type peptide (MAHHEEQHG-NH2), at a Cu(II)/ligand ratio of about 2:1 and in alkaline solution, a copper ion is bound to the N-terminal ATCUN site and the second one is instead anchored to the histidine residue in position 8 in the C-terminal domain (see Figure 1.). If Cu(II) and Ni(II) are simultaneously present in solution, the formation of ternary complexes is observed. [1] S.R. Hennigar, J.P. McClung, Am. J. Lifestyle Med., 2016, 10(3), 170–173. [2] D. Witkowska, S. Bielinska, W. Kamysz, H. Kozlowski, J. Bioinorg. Chem., 2011, 105(2), 208-214. [3] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Coord. Chem. Rev., 1999, 184, 311-318. [4] P. Gans, B. O'Sullivan, Talanta, 2000, 51, 33-37. [5] P. Gans, A. Sabatini, A. Vacca, Talanta, 1996, 43, 1739-1753.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
