unexpected impact of the number of glutamine residues on metal complex stability

The emerging question which this study aims to answer is: what impact do glutamines have on the stability of metal–peptide complexes? We focused our attention on the N-terminal domain of Hpn and Hpn-like proteins from Helicobacter pylori. Cu2+ and Ni2+ complexes of the model peptides MAHHE-NH2, MAHHEEQ-NH2, MAHHEQQ-NH2 and MAHHEQQHQA-NH2 were studied by means of different thermodynamic and spectroscopic techniques, as well as through molecular modelling computation. Experimental results, in very good agreement with theoretical findings, lead to the not obvious conclusion that the stability of metal complexes distinctly increases with the number of glutamine residues present in the peptide, although glutamine side-chains do not directly take part in coordination. This peculiar finding allows one to look at polyglutamine sequences, not only the ones present in some bacterial chaperones but also those involved in several neurodegenerative diseases, from a new perspective

Hexaamminecobalt(III) - Probing metal ion binding sites in nucleic acids by NMR spectroscopy

Hexaamminecobalt(III), an octahedral, inert metal ion complex, has lately gained increasing attention of structural biologists and bioinorganic chemists due to its use in structure determination of nucleic acids. This complex mimics outer-sphere binding events of the physiologically relevant magnesium(II)hexaaqua ion; hexaamminecobalt(III) often finds usage either in NMR spectroscopy, where cross-peaks between the complex ammines and the nucleic acid protons near the binding site are observed, in X-ray spectroscopy as heavy metal derivative for phasing, or in other techniques. In this review, we discuss the basic hexaamminecobalt(III) binding modes and give an overview on the most recent findings on [Co(NH3)6]Cl3–nucleic acid complexes. The various techniques that are applied in combination with this complex are mentioned and briefly summarized. Special attention is given to the application of [Co(NH3)6]Cl3 in nuclear magnetic resonance spectroscopy of nucleic acids, where it is used to reveal potential outer-sphere magnesium binding sites

Thermodynamic and spectroscopic study of Cu(ii) and Zn(ii) complexes with the (148-156) peptide fragment of C4YJH2, a putative metal transporter of: Candida albicans

Candida albicans is a widespread human pathogen which can infect humans at different levels. Like the majority of microorganisms, it needs transition metals as micronutrients for its subsistence. In order to acquire these nutrients from the host, C. albicans employs various strategies, also involving chelating proteins specifically expressed to sequester metals from the environment. A histidine-rich protein sequence identified in the C. albicans genome, named C4YJH2, has been recently studied for its putative role in Zn(ii) transport. Two outer membrane major histidine-rich clusters of C4YJH2, namely the domains 131-148 (FHEHGHSHSHGSGGGGGG) and 157-165 (SHSHSHSHS), have been confirmed as strong binding sites for the Cu(ii) and Zn(ii) ions. Nevertheless, the 9-residue "linker" sequence 148-156 (GSDHSGDSK) between the two His-rich fragments of C4YJH2, containing an additional His residue, can also contribute to metal binding. In the present work, the protected peptide Ac-GSDHSGDSK-NH2 and some analogues (Ac-GSDHSGASK-NH2, Ac-GADHAGDAK-NH2, Ac-GSDH-NH2, and Ac-HSGD-NH2) have been synthesized and their metal binding properties have been studied in detail. The thermodynamics of complex-formation equilibria of the above reported ligands with Cu(ii) and Zn(ii) ions have been studied by potentiometry in a wide pH range and the stoichiometry of the formed species has been confirmed by mass spectrometry; the most likely solution structures of the metal complexes are also discussed on the basis of NMR, UV-vis, circular dichroism (CD) and EPR data. The results show the importance of Asp7 in the stabilization of Zn(ii) complexes and suggest a significant role of the (quite abundant) Ser residues in the task of metal uptake and regulation

His-rich sequences – is plagiarism from nature a good idea?

In chemistry, nature – inspired solutions are often the most trivial and effective ones. Histidine rich sequences are used commercially in immobilized metal affinity chromatography (IMAC) as molecular ‘anchors’ that bind to a metal ion (usually nickel), immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support. The typical (His)6 tag, present at the C- or N- terminus of a protein which is meant to be purified, has been successfully used for decades. Consecutive histidines are the common denominator for both His-tags used in molecular biology and for quite remote biological phenomena – polyhistidine sequences are found in some bacterial chaperons, in Zn2+ transporters, prion proteins, in histidine-rich glycoproteines (HRG), which posess a massive amount of functions, in some snake venoms and antimicrobal peptides. This work debates on two questions – first, why were such sequences chosen by nature to exist in some parts of specific, sometimes evolutionally remote proteins, and second, are we right about choosing the polyhistydyl motif as a perfect metal binder

Specific poly-histidyl and poly-cysteil protein sites involved in Ni(2+) homeostasis in Helicobacter pylori. Impact of Bi(3+) ions on Ni(2+) binding to proteins. Structural and thermodynamic aspects

Cysteine and histidine residues are tempting donors for Ni(2+) which coordinates to the sulfur of Cys and amide nitrogen atoms, or, in the absence of available thiol groups, to His imidazoles and amides. Bi(3+), on the other hand, has a very strong affinity towards Cys thiol groups, and can also coordinate an additional His imidazole. In this review, the complicated pathway of nickel uptake, delivery and regulation in microorganisms is summarized. We show potential binding sites, binding geometries, protein structures and discuss the predicted thermodynamic and kinetic aspects. We focus on the numerous recent observations on the homeostasis of nickel in Helicobacter pylori (H. pylori), a Gram-negative bacterium that colonizes the gastric mucosa in humans, and is the causative agent of acute and chronic gastritis. peptic ulcer disease, gastric carcinoma, and gastric lymphoma. The homeostasis of Ni(2+) is crucial for the survival of H. pylori in the extremely acidic environment of the stomach. The metal is delivered to urease (which catalyzes the hydrolysis of urea into carbon dioxide and ammonia and therefore neutralizes the low gastric pH) and to hydrogenase (which permits respiratory based energy production for the bacteria in the mucosa) by a set of accessory proteins. Most of the bacterium's metal metabolism is centered upon their expression and maturation. Below, a detailed description of the structural and thermodynamic aspects of the binding of nickel ions to poly-histidyl and poly-cysteil sites of urease and hydrogenase accessory proteins is given. Because bismuth compounds are one of the treatments for peptic ulcer disease, the inhibitory effect of Bi(3+) ions is described; the affinity of bismuth towards Cys side groups is much stronger than the affinity of nickel towards the same sites, therefore bismuth is able to displace nickel from its binding site, causing the inhibition of nickel chaperones

Specific interactions of Bi(III) with Cys-Xaa-Cys unit of a peptide sequence

The medicinal application of bismuth compounds is focused in two fields: antimicrobial and anticancer. Bi(III) complexes have been used in medicine as an effective treatment of microbial infections, such as peptic ulcers, diarrhoea, gastritis and syphilis. 212Bi and 213Bi are strong a-particle emitters, which, bound to specific ligands, could be promising targeted radio-therapeutic agents for the treatment of cancer. In this work, the coordination of bismuth to three peptides with the Cys-Xaa-Cys motif was studied by potentiometric, spectroscopic, mass spectrometric and NMR methods. We have shown, that sulfur atoms from cysteines are critical donors for the coordination of Bi(III). Our investigation provides insight towards an understanding of the chemistry of bismuth-containing complexes and may lead to the further application of this metal in medicine

Poly-His and poly-Gln sequences in bacterial proteins: tempting sites for metal ions to interact with

Hpn and Hpn-like are Helicobacter pylori cytoplasmic proteins involved in the homeostasis of nickel, required for the metal-enzymes urease and Ni-Fe hydrogenase, essential for the bacterium colonization in the human stomach. Hpn is an amazingly peculiar protein: almost half of its sequence consists of polyhistydyl repeats. On the other hand, Hpn-like proteins are rich in glutamine residues. Our research group was recently involved in a study on the Ni(II) and Cu(II) complexes of different fragments and analogues of Hpn and Hpn-like proteins, in order to shed light on the role of the consecutive His and Gln residues in metal-ion binding [1,2] (see Figure). The encouraging results pushed us to continue this investigation, focusing the attention on the N-terminal domain of Hpn-like proteins. Cu(II) and Ni(II) complexes of peptide models (MAHHE-NH2, MAHHEEQ-NH2, MAHHEQQ-NH2 and MAHHEQQHQA–NH2) were studied by means of different thermodynamic and spectroscopic techniques, as well as through molecular modeling computations

Metal specificity of the Ni(ii) and Zn(ii) binding sites of the N-terminal and G-domain ofE. coliHypB

HypB is one of the chaperones required for proper nickel insertion into [NiFe]-hydrogenase.Escherichia coliHypB has two potential Ni(ii) and Zn(ii) binding sites—the N-terminal one and the so-called GTPase one. The metal-loaded HypB-SlyD metallochaperone complex activates nickel release from the N-terminal HypB site. In this work, we focus on the metal selectivity of the two HypB metal binding sites and show that (i) the N-terminal region binds Zn(ii) and Ni(ii) ions with higher affinity than the G-domain and (ii) the lower affinity G domain binds Zn(ii) more effectively than Ni(ii). In addition, the high affinity N-terminal domain, both in water and membrane mimicking SDS solution, has a larger affinity towards Zn(ii) than Ni(ii), while an opposite situation is observed at basic pH; at pH 7.4, the affinity of this region towards both metals is almost the same. The N-terminal HypB region is also more effective in Ni(ii) binding than the previously studied SlyD metal binding regions. Considering that the nickel chaperone SlyD activates the release of nickel and blocks the release of zinc from the N-terminal high-affinity metal site of HypB, we may speculate that such pH-dependent metal affinity might modulate HypB interactions with SlyD, being dependent on both pH and the protein's metal status

The coordination of Ni2+ and Cu2+ ions to polyhistidyl motif of Hpn protein: is it as strong as we think it is?

Hpn, one of Helicobacter pylori’s nickel accessory proteins, is an amazingly peculiar protein – almost half of its sequence consists of polyhistydyl residues. In this work, we try to understand the origin of this naturally occurring sequence, shedding some light upon the bioinorganic chemistry of Hpn’s numerous poly-His repeats. Using potentiometric, mass spectrometric and various spectroscopic techniques, we studied the Ni2+ and Cu2+complexes of the wild type Ac-THHHHYHGG-NH2 fragment of Hpn and of its six analogues, in which consecutive residues (His or Tyr) were replaced by Ala (Ala-substitution or Ala-scan approach), resulting in Ac-TAHHHYHGG-NH2, Ac-THAHHYHGG-NH2, Ac-THHAHYHGG-NH2, Ac-THHHAYHGG-NH2, Ac-THHHHAHGG-NH2 and Ac-THHHHYAGG-NH2 peptides, respectively. We found that the His-4 residue is critical for both Ni2+ and Cu2+ ion binding and the effectiveness of binding varies even if the substituted amino acid doesn’t take part in the direct binding