DOI: 10.1002/chem.201200780 The Coordination of Ni II and Cu II Ions to the Polyhistidyl Motif of Hpn Protein: Is It as Strong as We Think? Danuta Witkowska, [a] Rossella Politano, [b] Magdalena Rowinska-Zyrek, [a] Remo Guerrini, [c] Maurizio Remelli,* [b] and Henryk Kozlowski* [a] Introduction A protein that was rich in histidyl residues was named Hpn because it was first identified in H. pylori and had an amaz- ing affinity for nickel ions. The sequence of Hpn consists of 60 amino acids, 28 of which (47%) are histidine residues. [1] The Hpn protein stores nickel in Helicobacter pylori, a human gastrointestinal pathogen that is involved in gastri- tis, duodenal ulcers, and in the formation of some kinds of gastric tumors. [2, 3] H. pyroli produces two enzymes, urease and Ni–Fe hydrogenase, which are essential for bacteria sur- vival in the human stomach. [4, 5] Ni II ions, which are a cofactor in these two enzymes, are crucial for their proper function- ing; however, at the same time, excess nickel ions can also be potentially toxic and can cause cellular damage. Recent studies have shown that the Hpn protein is an important factor in nickel (and other metal)-storage and detoxifica- tion. [6, 7] This unstructured polypeptide is abundant in the cy- toplasm of H. pylori and accounts for approximately 2 % of all proteins that are synthesized by bacteria. Hpn mostly exists as a multimer in solution, with each monomer (7 kDa) reversibly binding five nickel ions at pH 7.4 (K d = 7.1 mm) and 8.5 equivalents of Cu II ions (K d = 2.16 mm). [8] The moder- ate K d value (7.1 mm) gives this protein the opportunity to facilitate nickel-transfer to H. pylori Ni-chaperones with lower K d values, such as HspA (K d = 1.1–1.8 mm). [9] Bacteria that lack Hpn, which are cultured in vitro, are more suscep- tible to nickel ions than the wild-type bacteria. Deletion of the hpn gene also increases the response to therapeutic forms of bismuth. [10] Hpn shows a change in conformation upon binding Ni II ions. [11] This protein is transcriptionally ac- tivated in the presence of Ni II ions by the nickel sensor NikR. [12] Our recent studies have shown that the N-terminal part of Hpn protein binds Ni II - and Cu II ions in the same way that native human albumin does, that is, it forms nickel- and copper complexes that involve a {NH 2 ,2 N  ,N im } donor set. [13] In addition, we have examined and described the coordina- tion ability of nickel and other metal ions towards two frag- ments with a Cys  Cys motif (Ac-CCSTSDSHHQ-NH 2 and Ac-EEGCCHGHHE-NH 2 ) on the C-terminal part of Hpn. These studies have shown that nickel ions form very stable planar complexes with both peptides, owing to the {2S  ,N  } binding mode. [14] Many of the proteins that are involved in Ni II homeostasis in bacteria contain a polyhistidyl motif in their sequence (e.g., HypB from B. japonicum or R. leguminosarum, SlyD from E. coli, and Hpn-like from H. pylori ). [15] Recombinant cells of Staphylococcus xylosus and S. carnosus were shown to gain Ni II -ion-binding capacity after they were genetically modified to express surface-exposed chimeric proteins that Abstract: Hpn, one of Helicobacter py- lori’s nickel-accessory proteins, is an amazingly peculiar protein: Almost half of its sequence consists of polyhis- tidyl (poly-His) residues. Herein, we try to understand the origin of this nat- urally occurring sequence, thereby shedding some light on the bioinor- ganic chemistry of Hpn’s numerous poly-His repeats. By using potentio- metric, mass spectrometric, and various spectroscopic techniques, we studied the Ni II - and Cu II complexes of the wild-type Ac-THHHHYHGG-NH 2 fragment of Hpn and of its six ana- logues, in which consecutive residues (His or Tyr) were replaced by Ala (Ala-substitution or Ala-scan ap- proaches), thereby resulting in Ac- TAHHHYHGG-NH 2 , Ac-THAH- HYHGG-NH 2 , Ac-THHAHYHGG- NH 2 , Ac-THHHAYHGG-NH 2 , Ac- THHHHAHGG-NH 2 , and Ac- THHHHYAGG-NH 2 peptides. We found that the His4 residue is critical for both Ni II - and Cu II -ion binding and the effectiveness of binding varies even if the substituted amino acid does not take part in the direct binding interac- tions. Keywords: coordination modes · copper · histidine · nickel · proteins [a] D. Witkowska, Dr. M. Rowinska-Zyrek, Prof. H. Kozlowski Faculty of Chemistry, University of Wroclaw F. Joliot-Curie 14, 50-383 Wroclaw (Poland) E-mail : henryk.kozlowski@chem.uni.wroc.pl [b] R. Politano, Prof. M. Remelli Department of Chemistry, University of Ferrara via L. Borsari 46, 44100 Ferrara (Italy) E-mail: rmm@unife.it [c] Prof. R. Guerrini Department of Pharmaceutical Sciences, University of Ferrara via Fossato di Mortara 17/19, 44121 Ferrara (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201200780.  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2012, 18, 11088 – 11099 11088