A strong 13C chemical shift signature provides the coordination mode of histidines in zinc-binding proteins

Zinc is the second most abundant metal ion incorporated in proteins, and is in many cases a crucial component of protein three-dimensional structures. Zinc ions are frequently coordinated by cysteine and histidine residues. Whereas cysteines bind to zinc via their unique S(γ) atom, histidines can coordinate zinc with two different coordination modes, either N(δ1) or N(ε2) is coordinating the zinc ion. The determination of this coordination mode is crucial for the accurate structure determination of a histidine-containing zinc-binding site by NMR. NMR chemical shifts contain a vast amount of information on local electronic and structural environments and surprisingly their utilization for the determination of the coordination mode of zinc-ligated histidines has been limited so far to (15)N nuclei. In the present report, we observed that the (13)C chemical shifts of aromatic carbons in zinc-ligated histidines represent a reliable signature of their coordination mode. Using a statistical analysis of (13)C chemical shifts, we show that (13)C(δ2) chemical shift is sensitive to the histidine coordination mode and that the chemical shift difference δ{(13)C(ε1)} - δ{(13)C(δ2)} provides a reference-independent marker of this coordination mode. The present approach allows the direct determination of the coordination mode of zinc-ligated histidines even with non-isotopically enriched protein samples and without any prior structural information.     

Trace metals in the brain: allosteric modulators of ligand-gated receptor channels,the case of ATP-gated P2X receptors

Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.     

Analysis of zinc binding sites in protein crystal structures

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.     

Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.     

Metals on the move: zinc ions in cellular regulation and in the coordination dynamics of zinc proteins

Homeostatic control maintains essential transition metal ions at characteristic cellular concentrations to support their physiological functions and to avoid adverse effects. Zinc is especially widely used as a catalytic or structural cofactor in about 3000 human zinc proteins. In addition, the homeostatic control of zinc in eukaryotic cells permits functions of zinc(II) ions in regulation and in paracrine and intracrine signaling. Zinc ions are released from proteins through ligand-centered reactions in zinc/thiolate coordination environments, and from stores in cellular organelles, where zinc transporters participate in zinc loading and release. Muffling reactions allow zinc ions to serve as signaling ions (second messengers) in the cytosol that is buffered to picomolar zinc ion concentrations at steady-state. Muffling includes zinc ion binding to metallothioneins, cellular translocations of metallothioneins, delivery of zinc ions to transporter proteins, and zinc ion fluxes through cellular membranes with the result of removing the additional zinc ions from the cytosol and restoring the steady-state. Targets of regulatory zinc ions are proteins with sites for transient zinc binding, such as membrane receptors, enzymes, protein–protein interactions, and sensor proteins that control gene expression. The generation, transmission, targets, and termination of zinc ion signals involve proteins that use coordination dynamics in the inner and outer ligand spheres to control metal ion association and dissociation. These new findings establish critically important functions of zinc ions and zinc metalloproteins in cellular control.     

Zinc fingers: DNA binding and protein-protein interactions

The zinc finger domain is a very ubiquitous structural element whose hallmark is the coordination of a zinc atom by several amino acid residues (cysteines and histidines, and occasionally aspartate and glutamate). These structural elements are associated with protein-nucleic acid recognition as well as protein-protein interactions. The purpose of this review is to examine recent data on the DNA and protein binding properties of a few zinc fingers whose three dimensional structure is known.     

EXAFS investigation of the active site of iron superoxide dismutase of Escherichia coli and Propionibacterium shermanii

The local structure of the iron site in ferric superoxide dismutase from P. shermanii was analyzed by X-ray absorption spectroscopy. The metal-ligand cluster of the enzyme is found to be similar to the crystallographically investigated ferric superoxide dismutase from E. coli. At pH 6.4 the enzyme is five-fold coordinated with three histidines, an aspartate and a water molecule. The average bond lengths between the metal and the histidines are about 2.10 Å, between metal and aspartate they are about 1.86 Å and between metal and water 1.96 Å. With an increase in pH a change in the coordination number from five to six is observed both in pre-edge peak and EXAFS spectra analysis. However, the bond lengths of the ligands do not change dramatically, they are conserved for the aspartate and increase slightly to 2.13 Å for the average metal - histidine distance at pH 9.3. The observation of the increase in coordination number is correlated with a decrease in enzymatic activity which occurs in the high pH range. The zinc EXAFS spectra of P. shermanii superoxide dismutase have shown that zinc can be incorporated in the active center instead of the iron.     

High potency zinc modulation of human P2X2 receptors and low potency zinc modulation of rat P2X2 receptors share a common molecular mechanism

Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2–100 μm, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 μm. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nm) to very low (>100 μm) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.     

Metal Binding Is Critical for the Folding and Function of Laminin Binding Protein,Lmb of Streptococcus agalactiae

Lmb is a 34 kDa laminin binding surface adhesin of Streptococcus agalactiae. The structure of Lmb reported by us recently has shown that it consists of a metal binding crevice, in which a zinc ion is coordinated to three highly conserved histidines. To elucidate the structural and functional significance of the metal ion in Lmb, these histidines have been mutated to alanine and single, double and triple mutants were generated. These mutations resulted in insolubility of the protein and revealed altered secondary and tertiary structures, as evidenced by circular dichroism and fluorescence spectroscopy studies. The mutations also significantly decreased the binding affinity of Lmb to laminin, implicating the role played by the metal binding residues in maintaining the correct conformation of the protein for its binding to laminin. A highly disordered loop, proposed to be crucial for metal acquisition in homologous structures, was deleted in Lmb by mutation (ΔLmb) and its crystal structure was solved at 2.6 Å. The ΔLmb structure was identical to the native Lmb structure with a bound zinc ion and exhibited laminin binding activity similar to wild type protein, suggesting that the loop might not have an important role in metal acquisition or adhesion in Lmb. Targeted mutations of histidine residues confirmed the importance of the zinc binding crevice for the structure and function of the Lmb adhesin.     

The active site structure of tetanus neurotoxin resolved by multiple scattering analysis in X-Ray absorption spectroscopy.

A detailed study of the x-ray absorption spectrum of tetanus neurotoxin in the K-edge EXAFS region of the zinc absorber is presented that allows the complete identification of the amino acid residues coordinated to the zinc active site. A very satisfactory interpretation of the experimental data can be given if multiple scattering contributions are included in the analysis. Comparing the absorption spectrum of tetanus neurotoxin to that of two other structurally similar zinc-endopeptidases, thermolysin and astacin, in which the zinc coordination mode is known from crystallographic data, we conclude that in tetanus neurotoxin, besides a water molecule, zinc is coordinated to two histidines and a tyrosine.     

Toward a property/function relationship for metallothioneins: histidine coordination and unusual cluster composition in a zinc-metallothionein from plants

Early cysteine labeled (E(C)) proteins are plant metallothioneins, which were first identified in wheat embryos and are thought to be seed-specific. An exhaustive analysis of expressed sequence tag (EST) entries reveals that homologs are expressed in embryos of both classes of flowering plants (monocotyledons and dicotyledons), but also occur in conifers (gymnosperms) and seed-free spike moss (lycophyta). Mass spectrometric and elemental analysis results indicate that, contrary to the widely propagated number of five, E(C) binds predominantly six zinc ions in at least two zinc-thiolate clusters. 1H and 111Cd NMR experiments suggest that, in contrast to the majority of previously characterized metallothioneins, two conserved histidine residues participate in metal binding. The collected data is consistent with the presence of clusters unprecedented in metallothioneins so far. This novel cluster composition is accompanied by metal-binding properties that are substantially different from other metallothioneins; thus wheat E(C) binds zinc less strongly than either mammalian or cyanobacterial MTs. The unique biochemical properties of wheat E(C) render it ideally suited for a role in zinc donation to nascent proteins during seed development, a role that has been suggested based on the fact that E(C) is induced by the plant hormone abscisic acid, but not by heavy metals. Our results provide a step further toward developing a property/function relationship for metallothioneins.     

Active-site modification of superoxide dismutase by H2O2 studied through 1H NMR of the cobalt derivatives

It is known that H2O2 at pH 10, inactivates copper(II)-zinc(II)-SOD although not much information is available on what happens at the ligands coordinated to the two metal ions. We have reinvestigated the system through the electronic and 1H NMR spectra of the cobalt(II) and copper(II)-cobalt(II) derivatives. Such studies indicate that the coordinated residues are maintained although there is evidence of some flexibility of the donor groups. The coordination around copper is slightly more tetragonal. Azide binding to the copper ion does not cause the complete detachment of one of the histidines from the copper coordination sphere, as happens with the untreated enzyme.     

The X-ray structure of the zinc transporter ZnuA from Salmonella enterica discloses a unique triad of zinc-coordinating histidines

ZnuA is the soluble component of the high-affinity ZnuABC zinc transporter belonging to the cluster 9 group of ATP-binding cassette-type periplasmic Zn- and Mn-binding proteins. In Gram-negative bacteria, the ZnuABC system is essential for zinc uptake and homeostasis and is an important determinant of bacterial resistance to the host defense mechanisms. The cluster 9 members share a two (α/β)₄ domain architecture with a long α-helix connecting the two domains. In the Zn-specific proteins, the so-called α3c and the α4 helices are separated by an insert of variable length, rich in histidine and negatively charged residues. This distinctive His-rich loop is proposed to play a role in the management of zinc also due to its location at the entrance of the metal binding site located at the domain interface. The known Synechocystis 6803 and Escherichia coli ZnuA structures show the same metal coordination involving three conserved histidines and a glutamic acid or a water molecule as fourth ligand. The structures of Salmonella enterica ZnuA, with a partially or fully occupied zinc binding site, and of a deletion mutant missing a large part of the His-rich loop revealed unexpected differences in the metal-coordinating ligands, as histidine 140 from the mobile (at the C-terminal) part of the loop substitutes the conserved histidine 60. This unforeseen coordination is rendered possible by the “open conformation” of the two domains. The possible structural determinants of these peculiarities and their functional relevance are discussed.     

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H(158)WHD(161). To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines 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. The results support an intermolecular exchange mechanism of zinc transport.     

Zinc coordination geometry and ligand binding affinity: the structural and kinetic analysis of the second-shell serine 228 residue and the methionine 180 residue of the aminopeptidase from Vibrio proteolyticus

The chemical properties of zinc make it an ideal metal to study the role of coordination strain in enzymatic rate enhancement. The zinc ion and the protein residues that are bound directly to the zinc ion represent a functional charge/dipole complex, and polarization of this complex, which translates to coordination distortion, may tune electrophilicity, and hence, reactivity. Conserved protein residues outside of the charge/dipole complex, such as second-shell residues, may play a role in supporting the electronic strain produced as a consequence of functional polarization. To test the correlation between charge/dipole polarity and ligand binding affinity, structure-function studies were carried out on the dizinc aminopeptidase from Vibrio proteolyticus. Alanine substitutions of S228 and M180 resulted in catalytically diminished enzymes whose crystal structures show very little change in the positions of the metal ions and the protein residues. However, more detailed inspections of the crystal structures show small positional changes that account for differences in the zinc ion coordination geometry. Measurements of the binding affinity of leucine phosphonic acid, a transition state analogue, and leucine, a product, show a correlation between coordination geometry and ligand binding affinity. These results suggest that the coordination number and polarity may tune the electrophilicity of zinc. This may have provided the evolving enzyme with the ability to discriminate between reaction coordinate species.     

Different redox states of metallothionein/thionein in biological tissue

Mammalian metallothioneins are redox-active metalloproteins. In the case of zinc metallothioneins, the redox activity resides in the cysteine sulfur ligands of zinc. Oxidation releases zinc, whereas reduction re-generates zinc-binding capacity. Attempts to demonstrate the presence of the apoprotein (thionein) and the oxidized protein (thionin) in tissues posed tremendous analytical challenges. One emerging strategy is differential chemical modification of cysteine residues in the protein. Chemical modification distinguishes three states of the cysteine ligands (reduced, oxidized and metal-bound) based on (i) quenched reactivity of the thiolates when bound to metal ions and restoration of thiol reactivity in the presence of metal-ion-chelating agents, and (ii) modification of free thiols with alkylating agents and subsequent reduction of disulfides to yield reactive thiols. Under normal physiological conditions, metallothionein exists in three states in rat liver and in cell lines. Ras-mediated oncogenic transformation of normal HOSE (human ovarian surface epithelial) cells induces oxidative stress and increases the amount of thionin and the availability of cellular zinc. These experiments support the notion that metallothionein is a dynamic protein in terms of its redox state and metal content and functions at a juncture of redox and zinc metabolism. Thus redox control of zinc availability from this protein establishes multiple methods of zinc-dependent cellular regulation, while the presence of both oxidized and reduced states of the apoprotein suggest that they serve as a redox couple, the generation of which is controlled by metal ion release from metallothionein.     

Purification and properties of plaice metallothionein, a cadmium-binding protein from the liver of the plaice (Pleuronectes platessa).

A low-molecular-weight protein induced in the liver of the plaice (Pleuronectes platessa) by exposure to cadmium was purified and characterized. It is closely similar to mammalian metallothioneins in all of its properties in that it is a single-chain cadmium-binding protein of approx. 7000 mol.wt. with a high cysteine content (31 mol%) and no aromatic amino acid residues. The thiol groups of the cysteine residues complex with the cadmium in a SH/Cd molar ratio of 3:1 and produce a characteristic absorption maximum at 250 nm. Unlike the mammalian metallothioneins, however, metal analyses reveal only traces of zinc and copper in addition to cadmium. The presence of carbohydrate previously assumed from a positive reaction with periodic acid/Schiff reagent has now been disproved, and the positive reaction attributed to interaction with the thiol groups in the protein.     

Mutational analysis reveals only one catalytic histidine in neutral endopeptidase ("enkephalinase").

Aside from serving as zinc ligands, kinetic data has implicated one or more additional histidines as catalytic residues in neutral endopeptidase ("enkephalinase") action. One of these histidines has previously been identified as histidine 704 (Bateman et al., J. Biol. Chem., 265:8365-8368, 1990). In order to determine whether a second histidine is involved in catalysis each of these residues not previously changed have been converted to glutamine by site directed mutagenesis. The resultant recombinant enzymes possess full catalytic activity indicating that histidine 704 is the only catalytic histidine in the enzyme.     

Metal poison inhibition of carbonic anhydrase

Carbonic anhydrase is inhibited by the “metal poison” cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X-ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr-199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO₂) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor. © 1993 Wiley-Liss, Inc.