Slawomir Potocki

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 chaperones, in Zn2+ transporters, prion proteins, in histidine-rich glycoproteins (HRG), which posses a massive amount of functions, in some snake venoms and antimicrobial 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 polyhistidine motif as the strongest metal binder?

General Aspects of Metal Toxicity

This review is focused on the general mechanisms of metal toxicity in humans. The possible and mainly confirmed mechanisms of their action are discussed. The metals are divided into four groups due to their toxic effects. First group comprises of metal ions acting as Fenton reaction catalyst mainly iron and copper. These types of metal ions participate in generation of the reactive oxygen species. Metals such as nickel, cadmium and chromium are considered as carcinogenic agents. Aluminum, lead and tin are involved in neurotoxicity. The representative of the last group is mercury, which may be considered as a generally toxic metal. Fenton reaction is a naturally occurring process producing most active oxygen species, hydroxyl radical: Fe(2+) + He2O2 ↔ Fe(3+) + OH(-) + OH(•) It is able to oxidize most of the biomolecules including DNA, proteins, lipids etc. The effect of toxicity depends on the damage of molecules i.e. production site of the hydroxyl radical. Chromium toxicity depends critically on its oxidation state. The most hazardous seems to be Cr(6+) (chromates) which are one of the strongest inorganic carcinogenic agents. Cr(6+) species act also as oxidative agents damaging among other nucleic acids. Redox inactive Al(3+), Cd(2+) or Hg(2+) may interfere with biology of other metal ions e.g. by occupying metal binding sites in biomolecules. All these aspects will be discussed in the review.

Metal binding ability of cysteine-rich peptide domain of ZIP13 Zn2+ ions transporter.

The coordination modes and thermodynamic stabilities of the complexes of the cysteine-rich N-terminal domain fragment of the ZIP13 zinc transporter (MPGCPCPGCG-NH(2)) with Zn(2+), Cd(2+), Bi(3+), and Ni(2+) have been studied by potentiometric, mass spectrometric, NMR, CD, and UV-vis spectroscopic methods. All of the studied metals had similar binding modes, with the three thiol sulfurs of cysteine residues involved in metal ion coordination. The stability of the complexes formed in solution changes in the series Bi(3+) ≫ Cd(2+) > Zn(2+) > Ni(2+), the strongest being for bismuth and the weakest for nickel. The N-terminal fragment of the human metalothionein-3 (MDPETCPCP-NH(2)) and unique histidine- and cysteine-rich domain of the C-terminus of Helicobacter pyroli HspA protein (Ac-ACCHDHKKH-NH(2)) have been chosen for the comparison studies. It confirmed indirectly which groups were the anchoring ones of ZIP13 domain. Experimental data from all of the used techniques and comparisons allowed us to propose possible coordination modes for all of the studied ZIP13 complexes.

The specificity of interaction of Zn2+, Ni2+ and Cu2+ ions with the histidine-rich domain of the TjZNT1 ZIP family transporter

The Zrt/Irt-like protein (ZIP) family contributes to the metal homeostasis by regulating the transport of divalent metal cations such as Fe(2+), Zn(2+), Mn(2+), Cd(2+) and sometimes even Cu(2+). Most ZIP members have a long variable loop between transmembrane domains (TMDs) III and IV; this region is predicted to be located in the cytoplasm and is postulated to be the metal ion binding site. In this study, we looked at the thermodynamic behavior and coordination chemistry of Zn(2+), Ni(2+) and Cu(2+) complexes with the histidine-rich domain, Ac-(185)RAHAAHHRHSH(195)-NH2 (HRD), from the yeast TjZNT1 protein, located between TMDs III and IV. The sequence is conserved also in higher species like Thlaspi japonicum. The stability of complexes increases in the series Ni(2+) < Zn(2+)≪ Cu(2+). The geometry of complexes is very different for each metal and in the case of Zn(2+) complexes, high specificity in binding is observed. Moreover, the stability of HRD-Cu(2+) complexes was compared with the five His residues containing peptide from Hpn protein (Helicobacter pylori). The results suggest a high ability of HRD in the binding of all three studied metals.

The extracellular loop of IRT1 ZIP protein — the chosen one for zinc?☆

Zinc complexes with the extracellular loop of IRT1 (iron-regulated transporter 1), a ZIP (ZRT/IRT - Related Protein) family protein from Arabidopsis thaliana, have been studied. This unstructured fragment is responsible for metal selectivity and is located between the II and III transmembrane domains of IRT1. Zinc complexes with the Ac-(95)MHVLPDSFEMLSSICLEENPWHK(117)-NH2 peptide (IRT1), revealed surprisingly high thermodynamic stability. Additionally, an N-terminal fragment of human/mouse ZIP 13 zinc transporter (MPGCPCPGCGMACPR-NH2, later called ZIP13+C), has been chosen for the thermodynamic stability comparison studies. The relative ZIP13+C stability has been shown using several Zn(2+) complexes with artificially arranged multi-cysteine sequences. An interesting coordination mode has been proposed for the IRT1-Zn(2+) complex, in which imidazoles from two histidines (His-96 and His-116), a cysteine thiolate (Cys-109) and one of a glutamic acid carboxyl group are involved. All data were collected using potentiometric, NMR and mass spectrometric methods.

Metal Transport and Homeostasis within the Human Body: Toxicity Associated with Transport Abnormalities

In this work, latest reports about metal toxicity, transport and homeostasis have been thoroughly described and discussed. Although diseases associated with transport and homeostasis abnormalities are those of great interest, still a variety of the phenomena associated with these processes are under debate. In this paper, we try to summarize the newest theses on this topic, presenting contradictory points of view. We focus on toxic and essential metal pathways crossing and try to follow the exact metal binding molecules within the body and provide insight into the transport mechanism. Special attention is given to the mechanism of action of lately investigated metal transporters.