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

Abstract

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?

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Introduction

In the scope of this perspectives paper, we try to discuss, or at least briefly mention, most of the best known His-rich proteins or peptides, focusing on the question why this amino acid has been chosen by nature to be highly abundant in particular proteins. Is it a matter of being a good metal binder, or is it rather the just-below-physiological pK of imidazole, which allows His to act as a pH-switch that makes this amino acid so unique? The phenomenon of the repeated histidine sequences fascinates both chemists and biologists. Herein, we try to see if we are getting anywhere near understanding why such sequences exist in nature.

The issues discussed in the paper logically divide it into several parts. One by one, we discuss the most important groups of proteins that contain His-repeats and try to find out, what might be the link between them, or what feature makes all of them need the repeated histidine residue. First, we take a closer look at several bacterial His-rich proteins, most of which are involved in the homeostasis of Ni²⁺ ions. Second, we discuss why this type of sequence is present in several Zn²⁺ transporters and prion proteins. Then, we take a long walk along the pdb database to find and briefly recall several groups of biological molecules that contain more His residues than an average protein does.

When talking about polyHis sequences, it is impossible not to mention the polyHis tag, one of the most popular ones used in molecular biology. In the last part of the paper, we lead a detailed discussion about the thermodynamics of coordination of metal ions to the various His-rich motifs – both the artificial tags and naturally occurring sequences. We compare several of them, focusing on the metal binding abilities of such sequences and comparing the strength of coordination of metal ions to the naturally occurring His-rich sequences and to the artificial His-tags used in protein purification.

Bacterial His-rich sequences

One of the first poly-His bacterial protein isolated with the help of a nickel-coated column was found in sera from patients infected with H. pylori – a human gastrointestinal pathogen involved in gastritis, duodenal ulcers, and some kinds of gastric cancers. The metabolism of this bacteria is centered upon two nickel-containing enzymes – urease and hydrogenase. H. pylori produces numerous proteins involved in the maturation of these enzymes, or in the storage and detoxification of nickel ions. Large efforts are made to understand how the protein networks involved in the nickel homeostasis function and which Ni-dependent chaperone is the leading player in the nickel homeostasis ‘game’. Hpn, a 60-amino acid long protein that forms multimers in solution (which account for 2% of total protein synthesized in the cell), has been proposed to play a role in nickel storage.¹ 47% of Hpn's sequence are histidine residues, and among them there are two stretches of 6 and 7 consecutive histidines. The same study showed that Hpn was equally distributed between membrane and soluble fractions inside the bacterial cell.² The in vivo affinity of this protein towards metal ions decreases in the following order: Ni²⁺ > Bi³⁺ > Cu²⁺ ≈ Zn²⁺.³ This rather surprising specificity towards metal ions might most probably be the result of some additional biological phenomena taking place inside the cell. The results suggest a primary role of Hpn in Ni²⁺ storage and homeostasis in H. pylori; it is also the key factor that protects the bacteria from high concentrations of other metals. The protein was found to bind five Ni²⁺ ions per monomer at pH 7.4, with a dissociation constant (K_d) of 7.1 μM in a study, in which the dissociation constants were determined by equilibrium dialysis.³ Ni²⁺ binding induces conformational changes within the protein, increasing β-sheet and reducing the α-helical content, from 22% to 37%, and 20% to 10%, respectively, as shown by CD-spectroscopy.⁴ In another study, Hpn was used as a metal probe inserted between the FRET (fluorescence resonance energy transfer) partners CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein). The results confirmed that the binding of nickel to Hpn leads to conformational changes (Fig. 1).⁵

Fig. 1 The binding of metals to Hpn inserted between the FRET partners, CFP and YFP, results in a conformational change which can be seen as a change of FRET signal. Adapted from ref. 5.

Another histidine-rich protein which was found in H. pylori, Hpn-like, has a similar sequence to Hpn, and possesses two additional glutamine-rich motifs. It binds two nickel ions per monomer with a K_d of 3.8 μM.⁶ The expression of this protein, like the expression of other nickel chaperones in Helicobacter, is upregulated by HpNikR in the presence of excess Ni²⁺.⁷ The increased growth of recombinant Escherichia coli cells with the hpnl gene compared to those without this gene suggests a protective role of Hpnl under higher concentrations of external nickel ions.⁶ Hpnl may play a role in storage or detoxification of excess nickel. In addition, the studies provided by Seshadri et al. prove that both Hpn and the Hpn-like play a role in cobalt and cadmium tolerance. The greatest metal tolerance was attributed to the Hpn-like protein.⁸

Although Hpn has numerous His residues and also two Cys-Cys motifs in its sequence, which can bind nickel ions with very high affinity (involving two thiols and the amide nitrogen situated between them in the binding),⁹ this protein is able to bind not more than five metal ions per monomer, as mentioned above. The possible anchoring sites for Hpn and Hpn-like are shown in Fig. 2A and B, respectively.

Fig. 2 The sequence of (A) Hpn and (B) Hpn-like proteins from Helicobacter pylori (strain 26695).

Both of the proteins posses Glu and Tyr (and 1 Lys in the case of Hpn-like) residues, which can serve as donors for metal ions, but it is well known that at physiological pH, the imidazole nitrogens of His residues are much better binding sites.^10–12 The metal-binding properties of Hpn-like were recently thoroughly investigated. One study says that the N-terminal part of Hpn-like binds nickel and copper ions with a higher affinity than that of the Hpn protein, and also with a higher affinity than the N-terminus of albumin, despite the binding mode being the same in all cases {NH₂, N⁻, N_im}.¹³ The study performed by Zeng et al. suggests that histidine residues in the N terminus are not involved in metal binding and that both of the Ni²⁺-binding sites are localized in the His-rich domain (₂₁QHHHHHHAHHHHYYGGEHHHHNA₄₃) of Hpnl (from H. pylori 11637 strain).¹⁴ In an alanine-substitution study, His-29 and His-31 were proved to be crucial residues for the Ni²⁺ binding. The same work showed that Hpn-like also binds Cu²⁺, Co²⁺ and Zn²⁺ with moderate affinities, but possesses a higher affinity towards nickel ions in vivo. The discussed data lead us to the conclusion that Helicobacter's His-rich proteins play dual roles, as Ni²⁺ storage and as metal detoxification sites, depending on the exogenous metal levels.

What about other bacterial histidine-rich proteins? There are over 250 proteins which share more than 40% homology with H. pylori's Hpn, and 135 of them are bacterial proteins (UniProt database); the list keeps growing. The functions of most of these proteins are not characterized, but what we can be certain of is their sequence – all of them are rich in histidine residues. The ones which are characterized can be divided into: (1) accessory proteins required for the biosynthesis of the nickel active site in enzymes, (2) metal cation membrane transporters and (3) GTPases involved in cobalamin synthesis.

One of the most interesting accessory proteins important for hydrogenase maturation is HypB, a GTPase. HypBs expressed by Rhizobium leguminosarum and B. japonicum contain histidine-rich regions at their N-termini and possess high nickel binding capacities.¹⁵ HypB from Bradyrhizobium japonicum is a dimer, capable of binding 18 divalent nickel ions¹⁶ and R. leguminosarum's HypB binds about four Ni²⁺ per monomer.¹⁷B. japonicum's HypB has an extremely histidine-rich region at the N terminus – the fragment from His-16 to His-54 (HAHDHHHDHGHDHDHGHDGHHHHHHGHDQDHHHHHDHAH) contains 24 histidine residues. Truncated HypB lacking 23 of 24 His residues in this region was not able to bind to the (Ni-charged) metal chelate column.¹⁵ The Ni²⁺ that enters the cell of B. japonicum is bound in a form which is inaccessible to apoenzymes and in a much larger quantity than currently necessary for the Ni-containing hydrogenase; it is stored in order to allow hydrogenase maturation in the absence of external nickel.¹⁸ Such specific, tight binding of the metal protects the cell from the toxic effect of Ni²⁺, and at the same time makes it accessible for apo-nickel enzymes in times of nickel deficiency.¹⁹

Various bacteria from the Enterobacteriaceae family express proteins with poly-histidine sequences at their N-termini, which are usually hydrogenase accessory or nickel incorporation proteins (Fig. 3). Most of the bacteria from this family belong to human, animal or plant pathogens, like Salmonella spp. (causes acute diarrheas), Yersinia spp.,²⁰P. mirabilis (infects urinary tract),²¹Dickeya dadantii (causes wilting in a wide range of plants),²² or Enterobacter cloacae (is sometimes associated with urinary tract and respiratory tract infections). HypB proteins from H. pylori and E. coli do not have poly-His motifs, but in both cases they are required for hydrogenase maturation and supported by the HypA protein in Hp²³ and by the histidine-rich SlyD in E. coli.²⁴ Interactions between nickel chaperones and metal ions have been recently reviewed.^10,25

Fig. 3 Sequence alignment of N-termini of proteins involved in [NiFe]-hydrogenase maturation from D. dadanti, E. cloacae, Y. frederiksenii, E. bacterium and P. mirabilis. UniProt numbers: D2BS19, E3G8Z1, C4SU25, ESYKP3, C2LNG8 respectively.

The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins and revealed that SlyD is a significant component of the hydrogenase metallocenter assembly pathway in E. coli.²⁶ This protein probably acts as an activator of nickel release from E. coli's HypB.²⁷ SlyD consists of two well-separated domains: the FKBP domain, which harbors the prolyl isomerase activity, and the insert-in-flap (IF) domain, which harbors the chaperone activity.²⁸ The PPIase (peptidyl-prolyl isomerase) activity of SlyD is not required for maturation of Escherichia coli's hydrogenase.²⁹ A proteomic study of SlyD indicated that it is a stress-responsive protein – a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition.³⁰ The unstructured C-terminal part of this protein is extremely rich in His residues and is capable of coordinating a range of transition metal ions such as Ni²⁺, Cu²⁺, Co²⁺ as well as Zn²⁺.³¹ Investigations (by means of potentiometric, MS and spectroscopic techniques) on the coordination ability of two histidine-rich peptides from the C-terminus of the SlyD showed that in the case of the chosen peptide fragments, only imidazole side chains of histidine residues are involved in metal binding at physiological pH.³² One of these fragments (Ac-GHGHDHGHEHG-NH₂) forms equimolar complexes with nickel and copper ions. On the contrary, Ac-AHGHVHGAHDHHHD-NH₂ binds 2 metal ions per molecule at a pH range of 5 to 7. At higher pH, one of the metal ions is released and the second is bound to subsequent amide nitrogens. If the binding is the same in the case of the full length protein, this mechanism is probably responsible for ‘taking over’ the nickel ions by SlyD from other nickel chaperones which release Ni²⁺ when pH decreases, as shown by Kalurachchi et al.³⁷ Some authors suggest that SlyD plays a more general role in cellular metal homeostasis, in analogy with the eukaryotic and cyanobacterial metallothioneins.^33,34

Recent studies show that SlyDs interact with the large subunit of [NiFe]-hydrogenase and the HypB–SlyD interaction is independent from the SlyD-hydrogenase one, so it is possible that two or more molecules of SlyD are involved in hydrogenase maturation in Escherichia coli.³⁵ One acts as a chaperone for the precursor enzyme and one is responsible for nickel delivery in association with HypB. The structure of SlyD from Thermus thermophilus (which lacks the putative metal binding site earlier proposed for the cysteine-rich and histidine-rich C-terminal sequence in E. coli SlyD) shows that nickel is bound via a conserved C-terminal His-Gly-His-Xaa-His motif.³⁶ It is also the His-rich sequence which binds zinc ions in the case of this protein.³⁶ To check whether a single metal-binding motif (present in the above-mentioned SlyD from T. thermophilus) can functionally replace the full-length domain, a truncation of E. coli's SlyD was generated – SlyD155. Ni²⁺ binding to SlyD155 was studied using isothermal titration calorimetry, NMR and ESI-MS measurements.³⁷ Results show that SlyD155 could interact with HypB, but nickel release from HypB was substantially slower than in the presence of full-length SlyD. Moreover, SlyD155 was able to bind only one nickel ion, which was bound in an octahedral geometry with at least two histidines as ligands.³⁷ In the case SlyD from H. pylori, the solution structure shows that it is the His- and Cys-rich C-terminus that both Ni²⁺ and Zn²⁺ bind to, with dissociation constants of 2.74 and 3.79 μM respectively.³⁸

Some of the histidine-rich proteins were proposed also to facilitate the insertion of nickel into urease – the first known nickel enzyme.^39,40 In a recent study of K. aerogenes, the UreABC–UreDFG–UreE super complex was isolated confirming the role of UreE during urease enzyme maturation.^41,42 UreE from K. aerogenes contains 10 His residues in the C-terminal 15 amino acid sequence and binds six nickel ions per dimer with a K_d of 9.6 ± 1.3 μM.⁴³ X-ray absorption and variable-temperature magnetic circular dichroism spectroscopies reveal that nickel is bound to 3–5 histidyl imidazole ligands in a pseudo-octahedral geometry. Cu²⁺, Co²⁺ and Zn²⁺ compete with Ni²⁺ in the binding.⁴⁴ Cells which lacked the histidine-rich region in UreE showed only 73% of urease activity, and the activity decreased even more in cells with the whole UreE deletion.⁴⁵K. aerogenes UreE possess a conserved His96, which is not important for nickel binding, but is critical for the functioning of UreE in the maturation of urease in vivo, as well as in vitro.^46,47 Surprisingly, the histidine-rich region of UreE was not required for protection against nickel toxicity, as the strain with truncated UreE showed growth identical to the strain with the wild-type UreE in a medium containing 3 mM NiCl₂.⁴⁶

The nickel-binding capacity of UreE depends on the homolog, due to variable numbers of metal-binding residues at the C-terminus⁴⁸ and other accessory proteins that modulate the UreE nickel delivery process and/or its interaction with apo-urease.⁴⁹ The multi-protein systems can possess the ability to bypass the thermodynamic limitations of individual factors.

H. pylori's UreE, which does not have a poly-His motif, is a dimer which binds only one Ni²⁺ ion. Interestingly, by the addition of a (His)₆-tag to the C-terminus of H. pylori's UreE, it is possible to increase its nickel binding capacity, which further leads to an increase in urease activity.⁵⁰ Also in this case (as in the HypB protein), some authors note that organisms possessing high-affinity nickel-uptake systems do not have nickel-binding motifs in UreE, but organisms that have no uptake systems have histidine-rich regions.¹⁸ This deduction can be a bit too far-reaching, since numerous exceptions can be found, but still, it is worth to keep this thought in mind. It is known that H. pylori has a high affinity Ni²⁺ transporter NixA (K_m around 11.3 nM).⁵¹ Despite this fact, Hp, as mentioned above, possess at least two histidine-rich proteins involved in the maturation of urease and hydrogenase. Also B. japonicum exhibits a high affinity, nickel-specific transport protein (HupN).⁵²

One of the nickel enzymes which enables bacteria to survive under unfavorable conditions, apart from urease and hydrogenase, is carbon monoxide dehydrogenase (CODH).⁵³ CooJ is one part of the three-protein complex involved in normal in vivo Ni²⁺ insertion into CODH. The 12.6 kDa CooJ chaperone contains a C-terminal region with 16 histidine residues in the final 34 amino acids. This protein binds 4 Ni²⁺ ions per monomer with a K_d of 4 μM.⁵⁴ The histidine-rich fragment of CooJ is similar to that of HypB, described above.

His-rich regions can also be found among membrane-bound proteins. The NrsD from Synechocystis sp. strain PCC 6803⁵⁵ and NreB from R. metallidurans⁵⁶ are suggested to be responsible for nickel resistance by an efflux mechanism. These proteins are predicted to contain 12 transmembrane helices and histidine-rich C-termini. In 2005, a gene from a similar (from a functional point of view) protein was identified in E.coli; the protein was called rcnA (resistance to cobalt and nickel).⁵⁷ RcnA has a remarkable histidine-rich region (amino acids 121 to 146) with 17 histidines, 3 aspartates, and 3 glutamates that are predicted to form a cytoplasmic loop. RcnA does not change the steady state Ni²⁺ concentration in cells cultivated at low nickel concentrations, but lack of RcnA clearly increases the cellular nickel content at high but non-toxic nickel concentrations.⁵⁸ This protein could potentially act as both a nickel chelator and a nickel exporter, but the mechanistic importance of the His-rich region in RcnA is not yet understood.⁵⁹ Recently, another histidine-rich protein, RcnA, was found in human pathogen – Salmonella enterica.⁶⁰ It possess 26 His residues in a 50 amino acid long fragment (HHHDHDHDHDHDHDHDHDHDHDHDHDHDHDHDHDHDHDHDHDHHGHIHPE). Relationships between NikR (nickel-responsive transcriptional regulator), RcnA and RcnR repressor were recently comprehensively reviewed.⁶¹ His-rich domains in proteins can have a variety of other functions, can interact with a variety of ligands such as haem, heparin, plasmin, and IgG to regulate critical biological processes,^62–64 (discussed in the following sections of the paper), but as far as His rich sequences in bacterial proteins are concerned, their main role seems to be in nickel homeostasis and other divalent metal detoxification.

Prion proteins and His-rich metal transporters

It might seem surprising, how evolutionarily distant are the proteins for the functioning of which the histidine residue is most important. Prion proteins (PrPs), cellular components mainly expressed in neurons,⁶⁵ are obviously not as extremely His-rich as the proteins mentioned previously, but still, the His residues are abundant, and most of all, crucial for the prion pathogenesis. PrPs and PrP-like proteins are strongly conserved within vertebrates and characterized by a flexible disordered N-terminus and a C-terminal globular domain consisting of three helices and a short antiparallel β-sheet.^66,67 Many sources report that they possess some particular regions with histidine residues involved in metals binding. The tandem repeat region of mammalian PrP (residues 60–91), consisting of four octarepeat units (PHGGGWGQ), tightly binds Cu²⁺.^68,69 The unstructured N-terminal region of avian PrP is also characterized by a repeat region (residues 53–94), consisting of a hexapeptide (PHNPGY) which is also able to bind copper efficiently.^70,71 Up to 4 Cu²⁺ ions may be bound to the octapeptide repeat region (OR) of mammalian PrP, and further 1–2 Cu²⁺ ions to the N-terminus.^72–75 The repeat domains of StPrP (similar to PrP) and zebrafish PrP-like proteins (zPrP) from fugu and zebrafish, encompassing residues 94–128 and 60–87, respectively, are formed by irregular repeat units, each containing two histidine residues (GHGYGVYGH and HXGHXG for fugu and zebrafish, respectively). It has been shown that these fragments can bind copper even more efficiently than the human repeat domain.⁷⁶ All the obtained results show that all the single repeat units from human, chicken and piscine PrP are able to bind a single Cu²⁺ ion; in all cases, the His imidazole ring acts as a copper anchoring site allowing the successive main chain amide nitrogen deprotonation.

Studies concerning peptides, with the hydrophobic region hPrP91–126, revealed two independent Cu²⁺ binding sites located at His96 and/or His111.^77–79 Model peptides containing those histidine residues have been widely studied in order to characterize the copper coordination sphere. Numerous investigations indicate that histidine imidazole acts as an anchoring site, allowing subsequent amide nitrogen deprotonation yielding to [N_im, 2N⁻, O] (with the oxygen from either an adjacent carbonyl group or a water molecule) or [N_im, 3N⁻] coordination mode at a pH around 6.5 and 8.5, respectively.^80,81 Zinc has also been associated with PrP physiology. Although zinc exhibits a weak binding affinity toward mammalian prion protein,⁸² it promotes intermolecular interactions.⁸³ As for hPrP, copper binding to zPrP is much more effective than binding of zinc. In fact, zPrP63–80 and zPrP63–87 fragments can bind Cu²⁺ by histidine donors [4N_im] and at higher pH by histidine and amide nitrogen atom donors [N_im, 3N⁻], whereas Zn²⁺ is bound by four imidazole groups [4N_im] only.⁸⁴

Multi-histidine sequences are frequently found in metal transport proteins, some of which are closely related to prion proteins. His-rich regions are expected to play a crucial role either in the metal transport process itself or in the selectivity and specificity towards various metal ions. The behavior of intra- or extracellular loops of transmembrane transport proteins is determined in the presence of different ions in the cytoplasm or extracellular lumen, which may affect their substrate specificity. In order to answer the question what is the mechanism of action of metal transporters, many kinds of approaches have been used so far, however always bearing in mind a very probable effectual impact of the histidine residues.

The ZIP family of proteins (SLC39A, Zrt and IRT like proteins) is a large group of metal transporters evolutionary related to prions⁸⁵ with multi-histidine sequences whose function is currently under debate. Those transporters are responsible for the increase of zinc concentration in the cytoplasm^86–88 and possess six to eight trans-membrane domains (TMDs) and extracellular/cytoplasmic histidine-rich loops between them (Fig. 4). The loops contain histidine-rich domains (HRDs) and are postulated to serve as metal ion binding sites.⁸⁹

Fig. 4 The scheme of ZIP 6 protein as a representative of the ZIP family.

Over the years, the mechanism of ZIP transporters and the function of their histidine rich sequences remained unknown. Experiments were carried out on simplified artificial models, as well as on prokaryotic and eukaryotic cells. What was always taken into account when ZIP proteins were investigated is that the structure of zinc transporters is highly conserved among different species⁹⁰ – that is why results of studies on simple organisms may shed a new light on the mechanism of action of those present in more complex species e.g. in mammals. Studies on the Arabidopsis membrane protein IRT1, a transporter of essential metals like iron, zinc and manganese, showed that exchanging one amino acid from the multi-histidine cytoplasmic or extracellular loops changes the substrate specificity e.g. the replacement of a glutamic acid residue at position 103 in the extracellular region between the II and III trans membrane domain in wild-type IRT1 with alanine, by selectively eliminating its ability to transport zinc. Another mutation in the histidine-rich loops, replacing the aspartic acid residues at either positions 100 or 136 with alanine, also increases IRT1 metal selectivity by eliminating transport of both iron and manganese. A number of other conserved residues in or near transmembrane domains appear to be essential for all transport functions.⁹¹ However, what is most interesting about studies concerning ZIP proteins is a frequently noticed correlation of their functioning with the presence/absence of histidine residues. The long variable loop between transmembrane domains III and IV is predicted to be located in the cytoplasm. Surprising is the fact that deletion of the His-rich domain in yeast cultures of TjZNT1 (Fig. 5) does not affect the Ni²⁺ tolerance ability of this protein – a ZIP transporter that confers high Ni²⁺ tolerance to yeast.⁹² Further studies showed the effect of the His-rich domain deletion on the ion transport ability of TjZNT1. The absence of HRD increases the specificity for Zn²⁺, but not for Cd²⁺. In addition, studies with yeast cultures engaging fluorescent methods (GFP protein) indicated that the His-rich domain-deleted mutants have the same localization of TjZNT1, when compared to wild type microorganisms. These results indicate that His-rich domains might be involved in the ion specificity of ZIP proteins.

Fig. 5 Histidine rich domain (HRD) of the yeast TjZNT1 protein.

On the other hand, replacement of all histidine residues with glutamine in the HRD of the yeast ZRT1 has no apparent effect on the transporter function, but it has been found to alter ZRT subcellular localization. Other studies, concerning hZIP1 (human zinc transporter ubiquitously expressed), were performed with the use of directed-mutagenesis to replace the loop histidines with alanines in the histidine-rich motif, located in the large loop of this transporter. The aim was to determine if this multi-histidine domain is involved in the transport of zinc. Results suggest that both investigated histidines (from the ₁₅₈HWHD₁₆₁ sequence) are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter, as has been reported for the ZRT1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. These results support an intermolecular exchange mechanism of zinc transport.⁹³ The role of histidine-rich sequences in ZIP proteins is not only limited to affect transport itself, but is also crucial in the aging and degradation process of the protein. Recent studies identified a cytoplasmic multi-histidine domain that was crucial for ubiquitin-dependent degradation of ZIP4 and protection against zinc toxicity.⁹⁴ However, this motif was dispensable for zinc-induced endocytosis. These findings indicate that ubiquitin-mediated degradation of the ZIP4 protein is critical for regulating zinc homeostasis in response to the upper tier of physiological zinc concentrations, via a process that is different from zinc-stimulated endocytosis.

Thermodynamic–calorimetric studies quantified the metal-binding affinity of IRT1's His-rich region (PHGHGHGHGP) of Arabidopsis thaliana.⁹⁵ It has been demonstrated that although substrates of IRT1 (Fe²⁺ as well as Mn²⁺, Co²⁺, Zn²⁺, and Cd²⁺) did not bind tightly, this His-rich sequence has a very high entropy-driven affinity for Fe³⁺, which is not transported by IRT1. These reports have proposed both positive and negative effects on the participation of the His-rich domain in metal binding, transport and protein degradation.

Histidine-rich sequences are not only present in ZIP metal transporters, they are also abundant in the cation diffusion facilitator (CDF) family⁹⁶ and ABC metal transporters.^97,98 A very interesting, however, sometimes contradictory thesis about the mechanism of action of those proteins has been proposed. It was shown that a CDF protein from Bacillus subtilis uses an antiporter mechanism, catalysing active efflux of zinc in exchange for K⁺ and H⁺.⁹⁹ A further step concerning those proteins was the study of zinc efflux across the plasma membrane of Escherichia coli. It was the first kinetic study of the purified and reconstituted ZitB (CDF family) by stopped-flow measurements and metal-sensitive fluorescent proteoliposomes. It was established that this transport is coupled to H⁺ rather than to K⁺ efflux.¹⁰⁰ Concerning the involvement and effect of histidine residues on the functioning of the ZitB transporter, experiments using Escherichia coli were performed engaging site-directed mutagenesis to elucidate the function of individual amino acid residues. Substitutions of several charged or polar residues, e.g. histidines, located in predicted transmembrane domains, result in the loss of the ZitB function. Crystallographic (2.9 Å resolution) and fluorescent (FRET) studies of the Escherichia Coli YiiP CDF zinc transporter (which has significant sequence homology to human ZnTs) revealed a richly charged multi-histidine homodimer stabilized by zinc binding.¹⁰¹ The YiiP CDF protein structure exhibits a Y-shaped dimeric architecture. Structural features support an auto-regulatory mechanism, by which cytoplasmic zinc is capable of switching on the activity of YiiP; by this mechanism the protein removes excess of zinc from the cytoplasm. Four zinc ions are bound per monomer. Zinc binding leads to a scissor-like conformational change that affects the orientation of the transmembrane helices and the coordination of zinc at the primary transport site. In this case, histidine residues seems to play a crucial role in stabilizing the protein binding site together with its functional structure.

Other His-rich sequences in proteins and peptides

Above, we discussed the His-rich proteins that are well described in the literature. As a general conclusion, one can say that their main role is the binding of metal ions. We are, naturally, aware, that this is not the only function that histidine has in nature. Apparently, there are numerous His-rich proteins and peptides, for which we cannot explain the fact why they are His-rich. Several examples are listed below.

1. His-rich proteins in nuclear speckles

In general, His-rich sequences can be divided into those in which the His residues are consecutive and those in which the histidines are numerous, but separated by other amino acids. A recent comprehensive analysis of human proteins, containing a series of consecutive histidine tracts, revealed that there are 86 proteins in the human genome that contain stretches of five or more consecutive histidines.¹⁰² Almost ¾ of them can be found in the nucleus, where they are involved in DNA- and RNA-related functions. Most of them accumulate in the nuclear subcompartment known as nuclear speckles and, what is interesting is that this localization is lost when the histidine repeat is deleted, proving that the His-repeats are crucial for localizing the proteins in nuclear speckles.

The pattern of His-repeats varies from simple amino acid runs (for example, HHHHHHHHHH in HOXA1) to complex repeats, such as HPSNHHHHHNHHSHKHSH in cyclin T1), suggesting that it is most likely not the number of His residues that is decisive for its functional role but rather, the spacing between residues may be important.

This His-rich targeting signal is most probably an interaction surface for some of the molecules present in the speckle. The exact mechanism of this targeting is not yet clear, but since it is the His repeat that is necessary for proper targeting, the basis of the process must be associated with the unique chemical properties of histidine. At this point of our discussion, it is already clear that this amino acid is an excellent metal binder, but it can also be versatile because of other features. For example, the imidazole side-chain allows it to shift from a neutral to positive charge in a pH-dependent fashion, a property that may have an impact on the binding capabilities of a His-stretch. Moreover, the presence of His in a β-strand provides a charge gradient that could mediate protein–protein or protein–DNA electrostatic interactions (an example of a His-stretch as a protein–protein interacting domain can be found in cyclin T1 when interacting with RNA polymerase II and granulin).¹⁰³ All of the above-mentioned mechanisms may partially contribute to the regulation of the binding properties of His-repeats.

2. His-rich glycoproteins

The name of another family of proteins we want to briefly mention explains why we recall them in this review – histidine-rich glycoproteins (HRGs), also known as histidine-proline-rich glycoproteins, interact with such a wide variety of protein targets that they have been called the Swiss Army knife of mammalian plasma by some authors.¹⁰⁴ Their multidomain structure allows them to interact with many ligands, such as heparin, plasminogen, fibrinogen, immunoglobulin G, C1q, heme and Zn²⁺. The ability of HRGs to interact with several ligands simultaneously suggests that they can function as adaptor molecules and regulate numerous important biological processes, such as immune complex/necrotic cell/pathogen clearance, cell adhesion, angiogenesis, coagulation, and fibrinolysis. To give an idea about how His-rich are some of the regions of the His-rich glycoproteins, we show a part of the human HRG sequence as an example: HPHKHHSHEQHPHGHHPHAHHPHEHDTHRQHPHGHHPHGHHPHGHHPHGHHPHGHHPHCHD (residues 350–410, Uniprot number P04196).

Very recently, it has been shown that one of the roles of HRGs is in blood clotting – mice and humans deficient in HRGs have shortened plasma clotting times. The authors show that HRG binds factor XIIa with high affinity, and this interaction is enhanced in the presence of Zn²⁺, but does not bind factors XII, XI, or XIa. This suggests that HRG modulates the intrinsic pathway of coagulation, especially in the vicinity of a thrombus, where platelet release of HRG and Zn²⁺ will promote this interaction.¹⁰⁵

Among all of the broad functions of HRGs, they are a key regulator of immunity and vascular biology. Also, there are some functional similarities between HRGs and other multifunctional plasma proteins, such as the C-reactive protein, C1q, β glycoprotein I, and thrombospondin-1.¹⁰⁰ Despite the knowledge about numerous interactions with different ligands, or about similarities with other proteins, a lot of physiological roles of HRGs are still not totally understood. Also, the question about the role of numerous His repeats present in those proteins remains open.

3. His-rich antimicrobial peptides

In general, antimicrobial peptides encompass a variety of structural and sequential motifs; most of them are α-helical, cationic and amphipathic, but there are numerous exceptions for this rule. Some have thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. Others can be classified as rich in a particular amino acid, including Pro-, Phe-, Arg-, or His-rich peptides.¹⁰⁶ The His-rich ones, most important for us in this discussion, are a really interesting group of antimicrobial peptides. Their net charge is pH dependent within the physiological range, and because of this, the two best known groups of antimicrobial peptides – clavanins from tunicates¹⁰⁷ and histatins from saliva¹⁰⁸ – show stronger activity at low pHs.

Clavanins are cationic, amphipathic peptides that consist of 23 amino acids. They are remarkably rich not only in histidines, but also in glycines and phenylalanines, as is seen from the primary structure of clavanin A (VFQFLGKIIHHVGNFVHGFSHVF). The repeated Gly, His, and Phe residues play important roles in the antimicrobial actions of wild-type clavanin A.^109,110

Antimicrobial and antifungal histatins found in saliva have been found to play a role in wound-closure.¹¹¹ These small, histidine-rich, cationic peptides, ranging in size from 7 to 38 amino acid residues in length, are secreted by the parotid and sub-mandibular salivary glands in humans and some higher primates. Histatins 1, 3 and 5 contain 38, 32 and 24 amino acid residues, respectively, and the sequence of the first 22 amino acids of each histatin is identical.¹¹² Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5. The same results indicate that zinc binding to histatin 5 involves His-15 present within the HEXXH zinc binding motif, and copper binding occurs within the N-terminal DSH ATCUN motif.¹¹³ Histatins are of large interest to the pharmaceutical sector, since they can be applied in situations where conventional drugs are of limited use due to the appearance of drug resistance. The fact that those small molecules are a normal component of human saliva indicates that they should be well tolerated when applied clinically to a range of acute and chronic oral fungal infections.¹¹⁴

Another histidine-rich amphipathic cationic peptide, LAH4, has antibiotic and DNA delivery capabilities. The interaction of peptides from this family with model membranes is strongly pH dependent; at neutral pH, the membrane disruption is weak, but at acidic pH, the peptides strongly disturb the anionic lipid component of bacterial membranes and cause bacterial lysis. The peptides are effective antibiotics at both pH 7.2 and pH 5.5, although the antibacterial activity is strongly affected by the change in pH. It is quite tempting to speculate about the biological significance of the phenomenon of pH activation. Histidines, having a pK_a of approximately 6.5 for the isolated amino acids, are largely unprotonated and uncharged at physiological pH but become protonated and cationic at acidic pH. pH-regulated “switches” can be responsible for restricting the antimicrobial activity of histidine-rich peptides to certain compartments of cellular environment (i.e., acidified phagosome) or certain tissues and organs.¹¹⁵

4. His-rich malaria proteins

Histidine-rich sequences are abundant in at least five malaria proteins (HRP1, HRP2, EMP1, EMP2, and EMP3), identified on the surface of the cytoskeleton of erythrocytes infected with Plasmodium falciparum; in some of them, up to 73% of the sequence are histidine residues.¹¹⁶ The most known one, HRP2, is a histidine- and alanine-rich protein, with numerous repeats of the sequences AHH and AHHAAD. The content of histidine, alanine and aspartic acid in HRP2 is 34%, 10%, and 10% respectively.¹¹⁷ As in the previously discussed cases, also in this one, the role of His-repeats remains unknown. Most likely, these proteins (with the exception of HRP2) have been associated with cytoadherence of P. falciparum infected red blood cells and rosetting and may play an important role in clogging of the post-capillary venules, which is one of the main causes of severe cerebral malaria. They may also participate in parasite mature stages evasion of the immune system and their subsequent destruction in the spleen.¹¹⁸

5. His-rich venoms

Another interesting occurrence of a His-rich sequence has lately been identified in snake venoms, namely those found in the rough scale bush viper (Atheris squamigera), the green bush viper (Atheris chlorechis) and the great lakes bush viper (Atheris nitschei). Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionisation tandem mass spectrometry (ESI-MS/MS) and Edman degradation proved the venom sequence to be EDDHHHHHHHHHGVGGGGGGGGGG.¹¹⁹

Investigations of nucleic acid databases showed similarities of this sequence to precursor sequences of bradykinin potentiating peptides and C-type natriuretic peptides, species that are capable of affecting the cardiovascular system by acting on specific metalloproteases and receptors. The mentioned poly-His and poly-Gly peptides found in Atheris venoms most likely also act on the cardiovascular system by inhibiting particular metalloproteases.

Thermodynamics of metal binding to His-rich sequences

It is well known that the imidazole nitrogen contained in the side group of histidine is a good binding site for the so-called “intermediate” metal ions, according to Pearson's classification. These metal ions, although they can form coordination bonds with other donor atoms, such as O and S, have a distinct preference for nitrogen. In addition, at physiological pH, the imidazole group of His, characterized by a pK value of about 6.5, is partially deprotonated, thus exerting a buffer action and being available for metal ion binding. Therefore, the presence of a His residue at any position of a peptide chain constitutes an excellent anchorage point for Cu²⁺, Zn²⁺ or Ni²⁺, provided that such a site is accessible, what normally occurs when the involved portion of chain is unstructured.

It is well documented that, in the case of Cu²⁺ and Ni²⁺, the presence of a metal ion bound to His promotes the deprotonation/coordination of amide nitrogens of the chain, up to the saturation of the available coordination positions of the metal. This deprotonation preferentially occurs in the N-terminal direction, leading to the formation of a six-membered ring. However, in special cases, e.g. in the occurrence of tertiary amide groups due to the presence of proline residues in the chain (as for the prion protein,¹²⁰ the deprotonation/coordination can proceed in the C-terminal direction, with the formation of a seven-membered ring).

The efficacy of His as the anchoring site has led us to think that the availability of more His residues in series can produce complexes that are more and more stable. This is certainly true for Zn²⁺: this metal ion is not able to deprotonate the amide nitrogens but it can bind to several His residues, if available, as in Cu/Zn-SOD.¹²¹ Even in the case of Cu²⁺ and Ni²⁺ (but also Co²⁺ and Fe³⁺), poly-His sequences lead to the formation of very stable complexes: this gave the impulse to a very efficient method of purification of proteins containing “His-handles”, either natural or synthetically added for the purpose (immobilized metal ion affinity chromatography, IMAC).¹²²

The most used His-tag sequence in IMAC is hexa-His (H₆) linked to the protein to be purified, preferentially at its N-or C-terminal, so that it can be easily removed after purification. The stationary phase classically employed in IMAC contains, as a coordination site, Ni²⁺ ions partially complexed with nitriloacetic acid (NTA)¹²³ bound to a support. As mentioned above, the method has long been used for purifying unmodified proteins containing His residues¹²⁴ or His-tagged recombinant proteins, as well as to study metal–protein affinities.¹²⁵

In fact, several naturally occurring proteins contain poly-His sequences of different lengths (see above) and this results in their high affinity, in vivo, to borderline metal ions, also suggesting a role in the homeostasis of these metals. The consecutive His-tags are generally not structured and this makes them more flexible for the formation of chelate complexes. However, also the His-tag with the His residues interspersed with other amino acids have a good affinity for metals.¹²⁶

The model proposed for the coordination system Ni²⁺–NTA is that of a nickel ion in an octahedral coordination, with four positions occupied by NTA and two free to bind His (Fig. 6).

Fig. 6 Model of the interaction between residues in the His tag and the metal ion in the tetradentate IMAC ligand (NTA). Adapted from ref. 118 with permission.

The mechanism of binding between Ni²⁺ ions bound to surfaces modified with NTA and His-tags of different lengths or combinations His/Ala has been studied by Knecht et al.¹²⁶: they have measured a value for the apparent dissociation constant (K_d) of 14 ± 1 nM, by means of the Surface Plasmon Resonance (SPR) technique. They also demonstrated that the preferred coordination geometry is the one that involves two His residues separated by either one or four other residues, while the elongation of the His sequence reduces the affinity, probably for entropic reasons (Fig. 7). Previous studies reported a K_d value two orders of magnitude higher (1 μM), at neutral pH.¹²⁷

Fig. 7 The overall binding process of H₆ to immobilized Ni²⁺–NTA at moderate concentrations. Adapted from ref. 126 with permission.

Since the environment used to measure K_d is rather different from that present in an IMAC column during separation, the value of the dissociation constant can only give a rough idea of the protein affinity to the support and therefore of the possibility to separate/purify it. In fact, K_d is a conditional constant, heavily dependent on experimental conditions used for its measurement, as already abundantly demonstrated:¹¹⁶ it is therefore common to find in the literature very different values of K_d, which formally refer to the same system.

The problem is intrinsic in the very definition of K_d. In the case of the interaction of a metal ion (M) with a peptide or protein (P), K_d is defined as the constant of the following dissociation equilibrium:

PM ⇆ P + M

where the charges have been omitted for simplicity. If the concentrations of reactants and products are measured in molarity, we have:
and K_d has the dimensions of a molarity.

This definition, although somewhat useful from an operational point of view, rarely represents the real situation: the stoichiometry of the species formed is often different from 1 : 1, depending on the metal-to-ligand ratio and concentration; moreover, the same coordination sites can give rise to complexes of different geometry depending on the experimental conditions, as is the case of the octarepeat domain of the prion protein that can bind up to four Cu²⁺ ions (Fig. 8).¹²⁸

Fig. 8 Distinct PrP octarepeat Cu²⁺ binding modes with three, two, or one coordinated His residues, respectively. The actual coordination mode depends on pH and/or metal-to-ligand ratio. Adapted from ref. 126 with permission.

As already described above, many natural proteins contain His sequences and proved to be very good binders for metal ions; among them, the Hpn protein consists of 60 amino acid residues of which 47% are histidines.¹²⁹ The affinity of Hpn and Hpn-like proteins for Cu²⁺, Zn²⁺ and Ni²⁺ was deeply investigated by many research groups by means of different experimental techniques. Dissociation constants available in the literature are reported in Tables 1 and 2, along with those of other His-rich proteins.

Table 1 Dissociation constant data for Ni²⁺ binding to some His-rich proteins

Protein	Bacterium	Experimental conditions	K d/μM	Ref.
HypB	Bradyrhizobium japonicum	Equilibrium dialysis: 2 ml of protein (5–6 μM) against 1000 ml of Tris acetate (50 mM) and NaCl (50 mM), pH 8.25, supplemented with appropriate amounts of Ni^2+, 48 h at room temperature	2.3	16
HypB	Rhizobium leguminosarum	Equilibrium dialysis: 0.26 ml of protein (2.5–5 μM) against “native buffer”, supplemented with appropriate amounts of Ni^2+, 16 h at 4 °C	2.5	17
UreE	Klebsiella aerogenes	Equilibrium dialysis: protein (2 μM) against Tris (50 mM) (pH 7.6) or HEPES (50 mM) (pH 7.2) or sodium phosphate (50 mM) (pH 7.2) and 0.5% NaCl, supplemented with appropriate amounts of Ni^2+, 3 h at room temperature	9.6	43
H144*UreE	Klebsiella aerogenes	ITC: metal ion titration into protein solution (10 μM), at 25 °C in 100 mM Tris buffer, 100 mM NaCl, pH 7.45	0.36	130
H144*UreE	Klebsiella aerogenes	ITC: metal ion titration into protein solution (10 μM), at 25 °C in 50 mM Tes buffer, 100 mM NaCl, pH 7.45	0.38	130
UreE	Klebsiella aerogenes	ITC: metal ion titration into protein solution (10 μM), at 25 °C in 100 mM Tris buffer, 100 mM NaCl, pH 7.45	1.0 10	130
UreE	Klebsiella aerogenes	ITC: metal ion titration into protein solution (10 μM), at 25 °C in 50 mM Tes buffer, 100 mM NaCl, pH 7.45	0.59 14	130
UreE	Bacillus pasteurii	ITC: metal ion titration into protein solution (10 μM), at 25 °C in 100 mM Tris buffer, 100 mM NaCl, pH 7.45	7.7 333	130
UreE	Bacillus pasteurii	ITC: metal ion titration into protein solution (10 μM), at 25 °C in 50 mM Tes buffer, 100 mM NaCl, pH 7.45	8.3 62	130
UreE	Bacillus pasteurii	Equilibrium dialysis: protein (5 μM) against standard buffer, 100 mM NaCl, supplemented with appropriate amounts of Ni^2+, 16 h at room temperature	17	131
UreE	Bacillus pasteurii	Centrifugal filtration trough a membrane: protein (40 μM) Tris (50 mM), pH 7.5, NaCl (0.5 M), supplemented with appropriate amounts of Ni^2+, overnight at 25 °C	35	132
CooJ	Rhodospirillum rubrum	Equilibrium dialysis: 0.3 ml of protein (5 μM) against 0.3 ml of Tris (50 mM), pH 8.0, supplemented with appropriate amounts of Ni^2+, 24 h at room temperature	4.3	133
SlyD155	Escherichia coli	ITC	0.065	37
HspA	Helicobacter pylori	Equilibrium dialysis: 0.1 ml filled with 30–100 μg of protein against 100 ml of HEPES (50 mM) and NaCl (100 mM), pH 7.2, supplemented with appropriate amounts of Ni^2+, overnight at 4 °C	1.8	134
HspA	Helicobacter pylori	Equilibrium dialysis: 0.3 ml of protein (20 μM) against 100 ml of Tris (50 mM) and NaCl (100 mM), pH 6.9, supplemented with appropriate amounts of Ni^2+, overnight at room temperature	1.1	135
Hpn	Helicobacter pylori	Equilibrium dialysis: 0.2 ml of protein (5 μM) against 1000 ml of HEPES (20 mM) and NaCl (100 mM), pH 7.4, supplemented with appropriate amounts of Ni^2+, overnight at 4 °C	7.1	4
Hpn-FRET	Helicobacter pylori	Fluorescence: protein (250 nM), citrate buffer	0.079	5
Hpnl	Helicobacter pylori	Equilibrium dialysis: 1 ml of protein (60 μM) against 500 ml of Tris buffer (20 mM), pH 7.5, supplemented with appropriate amounts of Ni^2+, overnight at 4 °C	3.8	6
Hpnl	Helicobacter pylori	ITC: metal ion titration into Hpnl at 37 °C in 20 mM Tris buffer, pH 7.4	4.8 53	14

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Table 2 Dissociation constant data for metal binding to some His-rich proteins. For experimental details see Table 1

Protein	Bacterium	Experimental conditions	K d Cu^2+/μM	K d Zn^2+/μM	K d Co^2+/μM	K d Bi^3+/μM	Ref.
H144*UreE	Klebsiella aerogenes	ITC, Tris buffer	2.2	7.7	—	—	130
H144*UreE	Klebsiella aerogenes	ITC, Tes buffer	0.83	—	—	—	130
UreE	Bacillus pasteurii	ITC, Tris buffer	—	102	—	—	130
Hpn	Helicobacter pylori	Equilibrium dialysis	2.16	19.3	—	11.1	4
Hpn-FRET	Helicobacter pylori	Fluorescence	—	0.001	—	61.9	5
Hpnl	Helicobacter pylori	Equilibrium dialysis	2.52	10.6	12.4	—	6
Hpnl	Helicobacter pylori	ITC	4.0	16.7	19.6	—	14

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A metal–ligand system is completely characterized when the speciation model, containing both the stoichiometry of the stability constants of the formed species, is known. If a system contains a metal ion (M), a ligand (L) and the proton (H), each complex-formation reaction can be described by the following general equation:

pM + qL + rH ⇆ M_pL_qH_r

The corresponding overall formation constant, under given temperature and ionic strength conditions (but independent of pH and component concentrations), is given by:
where charges and (eventually) coordinated solvent molecules are omitted for clarity. The square bracketed terms are the molar concentrations of the complex and of the free components; the coefficient r is negative when the number of protons dissociated from the ligand L exceeds the maximum number of protons dissociated in the absence of metal ions, or in the case of hydroxylated species.

Due to the high number of acid/basic sites normally present in a protein it is almost impossible to determine the complete speciation model for a metal–protein system. Nonetheless, if the binding sites are known and the corresponding protein domain is unstructured, it is possible to study (relatively) short peptide models, containing all the relevant residues for complex formation. This type of study can be performed by potentiometry, calorimetry and/or various spectroscopic techniques, thus obtaining a detailed and complete picture of the stability and solution structure of the complexes formed. By means of suitable computer programs it is then possible to compute distribution and competition diagrams.

Witkowska and coworkers¹³ have studied the formation of Cu²⁺ and Ni²⁺ complexes with peptides corresponding to the N-terminal domains of Hpn and Hpnl, protected at the C-terminal. This study has confirmed that the N-terminal sequence (MAH-), which is of albumin-like type (ATCUN motif), is a coordination site very efficient for both metals. Although the coordination geometry and the donor-atom set is the same for the two peptides corresponding to the N-terminal domains of Hpn and Hpnl, the second one is a stronger ligand than the first, in agreement with the K_d values measured for the whole protein. The reason for this difference lies in the subsequent amino acid sequence and, in particular, the presence of a long series of Gln residues.

What might be useful when discussing the coordination of typical His-rich sequences towards Ni²⁺ or Cu²⁺ ions is the comparison of the binding ability of the ATCUN domain of Hpn (MAHHEEQHG-NH₂)¹³ towards Ni²⁺ or Cu²⁺ ions with that of a typical His-rich sequence derived from the center of the protein (e.g. Ac-THHHHHYHGG-NH₂¹³⁶) and thus far from the terminal amino group. This comparison can be performed by means of competition plots, which can be computed from the speciation models of the systems to be compared. In Fig. 9, the hypothetical situation is reported, in which equal concentrations of the two peptides compete for the metal ion, in a wide pH range. The observed trend is that normally found in similar cases: the protected His-rich fragment is able to bind the metal ion (either Cu²⁺ on Ni²⁺) at lower pH than the N-terminal domain. In the reported example, Ac-THHHHHYHGG-NH₂ forms more stable complexes than MAHHEEQHG-NH₂ up to pH 5.8 for Cu²⁺ and 6.5 for Ni²⁺. At higher pH values (and thus also at physiological pH), where the amide nitrogens take part in the coordination, the opposite behavior is observed – the polyHis sequences are no longer the strongest metal binders.

Fig. 9 Competition plots of600 dpi in TIF format)[QUESTION MARK]?> Ac-THHHHHYHGG-NH₂ and MAHHEEQHG-NH₂ with Cu²⁺ (top) and Ni²⁺ (bottom).^13,136 Simulated concentrations of metal and both peptides are all 0.001 M.

Conclusions and perspectives

The deeper we go discussing His-rich sequences, the more interesting the subject becomes and more questions seem to appear. What we can be certain of are three facts: first, His-rich sequences are present in proteins involved in metal transport and homeostasis – the imidazole nitrogen is an excellent metal binder for several biologically important transition metal ions, such as Cu²⁺, Ni²⁺ (in the case of bacteria) or Zn²⁺.

Second, most proteins with consecutive histidines are present in nuclear speckles. In this, and in many other cases, we do not know why nature has chosen this particular type of amino acid repeats to be present in these proteins.

Third, the coordination of transition metal ions to sequences with consecutive histidines is not as thermodynamically stable as one would expect. A detailed analysis shows that these types of peptides are excellent metal binders only in a narrow pH range. This is most probably advantageous for IMAC, since the proteins that are bound to nickel (immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support) through a (His)₆ tag are obviously meant to be washed out from the column, so the metal ion binding cannot be too stable from the thermodynamic point of view.

As for the two questions asked at the beginning of this paper, we can conclude that His-rich sequences were chosen by nature mostly because of their ability to bind metal ions, but this is not the only function they can possess. We are close to understanding the metal binding to bacterial chaperones, metal transporters, prion proteins and snake venoms; the exact role of the histidines in proteins present in nuclear speckles, in histidine-rich glycoproteins or antimicrobial peptides remains less understood, although we have the general knowledge that they may participate in cytoadherence, membrane disruption or protein signalling.

As far as artificial His-tags are concerned, then yes, we were right about choosing them as a metal binder in IMAC. However, one has to keep in mind that such types of sequences with consecutive His stretches are not the ones that bind transition metal ions with most affinity throughout the whole pH range.

Acknowledgements

This work was supported by the Polish Ministry of Science and Higher Education (KBN N N204 146537). M. Rowinska-Zyrek is supported by a scholarship from the Foundation for Polish Science.

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Footnote

† This article is included in the All Aboard 2013 themed issue.

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