Zn Binding Sites Research Articles

Conserved motif CMLD in silicic acid transport proteins of diatoms

Sequenced fragments of genes coding for silicon transporters (SITs) were analyzed for diatoms of evolutionarily distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kutzing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate Cylindrotheca fusiformis Reimann et Lewin with a keeled raphe system). SITs were found to contain a conserved motif, CMLD. Hydropathy profiles showed that the motif CMLD is between two transmembrane domains lacking Lys and Arg, and the domains were consequently assumed to play a role in the formation of a channel mediating silicic acid transport. The motif CMLD proved to be rare. Since Zn2+ is necessary for silica incorporation into diatom cells, a hypothesis was advanced that the motif CMLD acts as a Zn-binding site. Diatom growth suppression was observed in the presence of the alkylating agent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonic acid (AEDANS), which does not penetrate into the cell. Cys of the motif CMLD was assumed to act as a target for AEDANS. Zinc ions inhibited Cys alkylation in the synthetic peptide NCMLDY, testifying to the above hypothesis.

Activation of sphingomyelinase from Bacillus cereus by Zn 2+ hitherto accepted as a strong inhibitor

Sphingomyelinase (SMase) from Bacillus cereus has been known to be activated by Mg 2+, Mn 2+, and Co 2+, but strongly inhibited by Zn 2+. In the present study, we investigated the effects of several kinds of metal ions on the catalytic activity of B. cereus SMase, and found that the activity was inhibited by Zn 2+ at its higher concentrations or at higher pH values, but unexpectedly activated at lower Zn 2+ concentrations or at lower pH values. This result indicates that SMase possesses at least two different binding sites for Zn 2+ and that the Zn 2+ binding to the high-affinity site can activate the enzyme, whereas the Zn 2+ binding to the low-affinity site can inactivate it. We also found that the binding of substrate to the enzyme was independent of the Zn 2+ binding to the high-affinity site, but was competitively inhibited by the Zn 2+ binding to the low-affinity site. The binding affinity of the metal ions to the site for activating the enzyme was determined to be in the rank-order of Mg 2+ = Co 2+ < Mn 2+ < Zn 2+. It was also demonstrated that these four metal ions competed with each other for the same binding site on the enzyme molecule.

The ADAMTS Proteases, Extracellular Matrix, and Vascular Disease

The importance of proteases as mediators of extracellular matrix (ECM) degradation and vascular cell phenotype in the pathogenesis of vascular disease is indisputable. In fact, excellent cases can be made for the matrix metalloproteinases (MMPs) (see review1), the serine proteases (see review2), and the cysteine and aspartic proteases (see review3) being involved in many of the events in vascular disease. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Jonsson-Rylander and her colleagues show us that ADAMTS1, a member of another family of proteases, is also involved.4 Descendants from the ADAM family of proteases, the ADAMTS members (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs), currently numbering 19, evolved as nonintegral membrane proteins that associate with the cell surface and ECM through specific protein domains (see reviews5,6). Like the ADAM family, the ADAMTS proteases are multidomain proteins with common structural motifs that include an N-terminal signal sequence, a prodomain, a catalytic domain with Zn binding site, an ancillary domain, a disintegrin-like domain, a central thrombospondin repeat domain (TSR), a cysteine rich domain, a cysteine free spacer domain, and usually one or more TSRs. Some of the ADAMTS members also contain unique C-terminal domains that can contain PLAC and/or CUB sequences. These proteases are synthesized as zymogens that undergo processing by convertases such as furin and by metalloproteinases such as MT-4 MMP.7,8 Like the MMPs, the ADAMTS members can be inhibited by tissue inhibitors of matrix metalloproteinases or TIMPs. For example TIMP-3 is a potent inhibitor of ADAMTS4 and 5.9 Other inhibitors exist as well. For example, C-terminal truncation of ADAMTS4 can be blocked by TIMP-1.7 Proteins that share structural homology to the ADAMTS members also may function as ADAMTS inhibitors. One example is papilin, a protein produced by the fruit …

Factors governing the protonation state of Zn-bound histidine in proteins: a DFT/CDM study.

We have performed systematic theoretical studies to elucidate the factors governing the His protonation/deprotonation state in Zn-binding sites, especially those containing the ubiquitous Zn-His-Asp/Glu triad. Specifically, we have addressed the following three questions: (1) How does the transfer of the Zn-bound His imidazole proton to the second-shell Asp/Glu carboxylate oxygen depend on the composition of the other first-shell ligands and the solvent accessibility of the metal-binding site? (2) Can any second-shell ligand with a proton acceptor group such as the backbone carbonyl oxygen also act as a proton acceptor? (3) What is the effect of the Asp/Glu in the Zn-His-Asp/Glu triad on the Zn-bound water protonation state? To address these questions, we used a combination of quantum mechanical and continuum dielectric methods to compute the free energies for deprotonating a Zn-bound imidazole/water in various Zn complexes. The calculations show that whether the Zn-bound His is protonated or deprotonated depends on (1) the solvent accessibility of the metal-binding site, and (2) the Lewis acid ability of Zn, which is indirectly determined by both the first- and the second-shell Zn ligands. The calculations also show that the effect of the Zn-His-Asp/Glu interaction on the nucleophilicity of the Zn-bound water depends on the solvent accessibility of the catalytic Zn site. Furthermore, they show that the Asp/Glu side chain in the Zn-His-Asp/Glu triad can increase the negative charge of its partner, His, and create an anionic hole that may stabilize a cation in buried cavities, provided that the Zn complex is cationic/neutral. The findings of this work are in accord with available experimental data.

Interactions of Zn(II) Ions with Three His-Containing Peptide Models of Histone H2A

The interactions of Zn(ll) ions with the blocked hexapeptide models -TESHHK-, -TASHHK- and -TEAHHK- of the -ESHH- motif of the C-terminal of historic H2A were studied by using potentiometric and IH-NMR techniques. The first step of these studies was to compare the pKa values of the two His residues inside each hexapeptide calculated by potentiometric or H-NMR titrations. Hereafter, the potentiometric titrations in the pH range 5 11 suggest the formation of several monomeric Zn(ll) complexes. It was found that all hexapeptides bind to Zn(ll) ions initially through both imidazole nitrogens in weakly acidic and neutral solutions forming slightly distorted octahedral complexes. At higher pH values, the combination of potentiometric titrations and one and two dimensional NMR suggested no amide coordination in the coordination sphere of Zn(II) ions. Obviously, these studies support that the -ESHH- sequence of histone H2A is a potential binding site for Zn(II) ions similarly with the Cu(II) and Ni(ll) ions, presented in previous papers.

Metal Binding and Selectivity in Zinc Proteins

AbstractZinc is one of the most abundant metals in living organisms and is an essential co‐factor of many metabolic enzymes and transcription factors. In this review, based on a recent body of theoretical and experimental results, the physical bases are delineated for the following aspects of zinc binding and selectivity in proteins: (1) What is the most thermodynamically preferable coordination geometry of a bidentate ligand (such as carboxylate) or Zn in Zn‐binding sites? (2) What is the protonation state of the Cys side chain in Cys4 zinc‐finger cores? (3) How does a protein select Zn from the mixture of ions in the surrounding fluids? (4) What is the role of the second shell in metal binding and selectivity? The key results are summarized and the physical basis and/or implications of the findings are discussed.

A simple tandem repeat polymorphism is present in the eighth intron of FLJ12960, a possible queuine salvage enzyme gene

The putative gene FLJ12960 was identified from sequence analysis of the human genome and predicted to be a member of the family of tRNA-guanine-transglycosylases for queuine biosynthesis by protein sequence similarity at the Zn-binding site (Locus Link #79691 at http://www.ncbi.nlm.gov/LocusLink/). FLJ12960 is immediately downstream of the Dopamine Receptor D3 ( DRD3) gene. A Simple Tandem Repeat (STR) polymorphism was identified in intron 8 of the FLJ12960 gene. Primers designed to amplify the CA repeat detect 16 alleles from 121 to 151 base pairs, with a heterozygosity of 0.74.

Contamination with nonessential metals from a solid-waste incinerator correlates with nutritional and immunological stress in prefledgling black kites ( Milvus migrans)

We assessed whether levels of heavy metals in blood have detrimental effects on the health of prefledgling black kites ( Milvus migrans) exposed to emissions from a solid waste incinerator near Madrid, central Spain. As health indexes, we considered body mass corrected by structural size as a general indicator of nutritional condition and white blood cell counts (especially the heterophil:lymphocyte ratio) as an indirect measure of immune function. Nutritional condition was positively related to levels of Zn, an essential element with a major role in many physiological pathways, while the opposite trend was found for the essential Cu, the concentration of which tends to increase with decreasing nutritional condition. We found an interaction between Zn and Cd, indicating that high levels of Zn produce smaller effects of Cd on body mass. Potential immunological stress, expressed as an increase in the heterophil:lymphocyte ratio, caused by the toxic Cd increased as levels of Cu decreased. Thus, nestlings with low levels of Cu and poor nutritional condition suffered more immunological stress due to Cd contamination than nestlings with higher levels of Cu. We also found a negative interaction between Zn and Cd, indicating a response of essential Zn to increasing levels of Cd, which may be indicative of the negative effects of Cd on the immune system. These results suggest that the participation of metallothioneins in detoxification and metal regulation may also indirectly enhance the adrenal stress response to contaminants, probably through a competition of Cd and Zn for Zn binding sites on metallothioneins, reducing the ability of Cd to stress the immune system.

First-second shell interactions in metal binding sites in proteins: a PDB survey and DFT/CDM calculations.

The role of the second shell in the process of metal binding and selectivity in metalloproteins has been elucidated by combining Protein Data Bank (PDB) surveys of Mg, Mn, Ca, and Zn binding sites with density functional theory/continuum dielectric methods (DFT/CDM). Peptide backbone groups were found to be the most common second-shell ligand in Mg, Mn, Ca, and Zn binding sites, followed (in decreasing order) by Asp/Glu, Lys/Arg, Asn/Gln, and Ser/Thr side chains. Aromatic oxygen- or nitrogen-containing side chains (Tyr, His, and Trp) and sulfur-containing side chains (Cys and Met) are seldom found in the second coordination layer. The backbone and Asn/Gln side chain are ubiquitous in the metal second coordination layer as their carbonyl oxygen and amide hydrogen can act as a hydrogen-bond acceptor and donor, respectively, and can therefore partner practically every first-shell ligand. The second most common outer-shell ligand, Asp/Glu, predominantly hydrogen bonds to a metal-bound water or Zn-bound histidine and polarizes the H-O or H-N bond. In certain cases, a second-shell Asp/Glu could affect the protonation state of the metal ligand. It could also energetically stabilize a positively charged metal complex more than a neutral ligand such as the backbone and Asn/Gln side chain. As for the first shell, the second shell is predicted to contribute to the metal selectivity of the binding site by discriminating between metal cations of different ionic radii and coordination geometries. The first-shell-second-shell interaction energies decay rapidly with increasing solvent exposure of the metal binding site. They are less favorable but are of the same order of magnitude as compared to the respective metal-first-shell interaction energies. Altogether, the results indicate that the structure and properties of the second shell are dictated by those of the first layer. The outer shell is apparently designed to stabilize/protect the inner-shell and complement/enhance its properties.

Crystal Structure of a Prostate Kallikrein Isolated from Stallion Seminal Plasma: A Homologue of Human PSA

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42 Å resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn 2+ and Hg 2+, two known PSA inhibitors.

Complexation properties of a new macrocyclic polyaminic ligand (L) containing amidic pendant arms: crystal structure of [PbL](ClO 4) 2

Synthesis and characterisation of the new macrocyclic ligand 1,7-dimethyl-4,10-di(methylcarbamoylmethy)-1,4,7,10-tetraazacyclododecane (L) are reported. The ligand, based on cyclen (1,4,7,10-tetraazacyclododecane), has been functionalised by the insertion of two methyl groups and two amidic pendant arms linked to the amine nitrogens. The interaction of L with H +, Na(I), Ca(II), Cu(II), Zn(II), Pb(II), and Gd(III) ions has been studied by potentiometric titrations, microcalorimetric and 1H NMR measurements in 0.1 mol dm −3 Me 4NCl aqueous solution at 298.1±0.1 K. The thermodynamic data suggest that the N 4 moiety is the binding site for Cu(II) and Zn(II), while in the case of Pb(II) also the pendant arms are coordinated to the metal ion. The crystal structure of [PbL](ClO 4) 2 (space group P2 1/ a, a=12.883(2) Å, b=12.259(3) Å, c=17.275(5) Å, β=108.65(2)°, V=2585.0(11) Å 3, Z=4, R=0.0660, R W 2 =0.1467) shows the metal ion hexa-coordinated by the four nitrogen atoms of the cyclic tetra-amine and by the two amidic oxygens of the pendant arms.

Disruption of zinc homeostasis in Alzheimer's disease

The basic hypothesis being tested is that, in Alzheimer's disease (AD), the delicate balance of brain Zn is disrupted and may play a role in the pathogenesis of neuron degeneration. Micro-PIXE measurements reveal a significant elevation of Zn in senile plaques (SP) in AD brain compared with adjacent neuropil and a significant increase in AD neuropil compared to control neuropil. The observation of elevated Zn in SP is of interest because the amyloid precursor protein contains a Zn binding site that may prevent normal cleavage leading to the generation of a toxic fragment of beta amyloid, the constituent of SP. The potential of using laser-ablation inductively coupled plasma mass spectrometry as a complimentary microprobe technique is also presented.

Interactions of Azodicarbonamide (ADA) Species with the Model Zinc Finger Site: Theoretical Support of the Zinc Finger Domain Destruction in the HIV-1 Nucleocapsid Protein (NCp7) by ADA

Interactions of azodicarbonamide (ADA) species with zinc-containing sites, which mimic the zinc finger domains of the HIV-1 nucleocapsid protein (NCp7), have been investigated by quantum chemistry tools in order to provide theoretical insights into recent experimental findings. In the latter, reactions of ADA with NCp7 lead to destabilization of the zinc coordination sphere followed by HIV inhibition. In this work, energy profiles for the interactions of neutral and protonated ADA species with the Zn binding site have been calculated at the density functional theory level in the B3LYP approximation. The calculations show that the ADA molecule, protonated at the central nitrogen atom, reacts without activation barrier with the model zinc binding site, destructing the metal coordination sphere, while interactions of other ADA species do not lead to modification of the metal site.

A New Insight into Metallothionein (MT) Classification and Evolution: THE IN VIVO AND IN VITRO METAL BINDING FEATURES OF HOMARUS AMERICANUS RECOMBINANT MT

We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc- and copper-supplemented medium was achieved for MTH, the two domains betabetaMTH and betaalphaMTH and three site-directed mutants, betabetaC9H, betaalphaC37H, and betaalphaE31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M(II)6-MTH for the entire MTH and M(II)3-betabetaMTH and M(II)3-betaalphaMTH for the independent domains fully validated our expression system. A low affinity binding site for a seventh Zn(II) in the in vivo synthesized MTH was located in the betaalpha domain. Additionally, minor M(II)4 species were found for each domain. Both single Cys to His mutations exhibited a similar reduction of their in vivo zinc binding ability but differed in their cadmium binding behavior when compared with the wild-type forms. Conversely, the double mutant showed an enhanced zinc and cadmium binding capacity. In vivo synthesis of MTH and of its independent domains in the presence of copper only afforded heterometallic copper-zinc species. These findings allow consideration of MTH as a zinc thionein and question the view of all crustacea MT structures as copper thioneins. Furthermore, a new approach for the evolutionary and functional classification of MT is proposed, based on the stoichiometry of metal-MT species and molecular phylogenetic analysis.

Dinucleotide Hydrolysis Promoted by Dinuclear Zn Complexes − The Effect of the Distance between Zn Ions in the Complexes on the Hydrolysis Rate

The rate of hydrolysis of an RNA dimer, uridylyl(3′→5′)uridine (UpU), promoted by dinuclear Zn complexes resulting from a series of ligands with differently spaced Zn ion binding sites, was examined. Although the ligands in the present study have in common two di-2-pyridylmethylamino moieties as the Zn binding sites, the activities of these ligands toward the hydrolysis were quite different. Our results suggest that the distance between metal ion coordination sites is an essential factor for the intrinsic activities of the dinuclear complexes.

Isolation and characterization of the Candida albicans MOT2 gene.

A putative Candida albicans homologue of Saccharomyces cerevisiae MOT2 (modulator of transcription) has been cloned and analyzed. A cDNA fragment corresponding to a portion of S. cerevisiae MOT2 was used to isolate a similar C. albicans gene (CaMOT2). CaMOT2 is comprised of two exons of 50 bp and 1,714 bp, respectively, with a single 82 bp intron located near the 5' end of the gene. The gene encodes a protein (CaMot2p) with an estimated mass of 67 kDa. The 5' region of the gene shows sequence homology with S. cerevisiae MOT2, whereas no significant similarity was observed in the 3' region. Similarly, the N-terminal portion of C. albicans Mot2p exhibits approximately 80% homology with S. cerevisiae Mot2p, while no significant homology to any known protein was observed in the carboxy-terminal half of the C. albicans protein. The N-terminal portion of CaMot2p contains a cysteine-rich domain (amino acids 18-62). The distribution of the cysteine residues identifies CaMot2p as a zinc-finger protein. The data suggest two potential Zn-binding sites, similar to the arrangement found in S. cerevisiae. Reverse-transcriptase polymerase chain reaction was used to compare the level of CaMOT2 expression between C. albicans grown in vitro and growth during in vivo infection in the rat model of oral candidiasis. The results showed CaMOT2 is down-regulated during growth in the rat oral cavity compared to in vitro culture. Although the function of C. albicans MOT2 has not been determined, comparison to S. cerevisiae MOT2 suggests the gene product may act as a general negative regulator.

Parallel ab initio and molecular mechanics investigation of polycoordinated Zn(II) complexes with model hard and soft ligands: Variations of binding energy and of its components with number and charges of ligands

In this study we compare the binding energies of polycoordinated complexes of Zn2+ within cavities composed of model “hard” (H2O, OH−) or “soft” (CH3SH, CH3S−) ligands. Ab initio supermolecule computations are performed at the HF and MP2 levels using extended basis sets to determine the binding energies and their components as a function of: the number of ligands, ranging from three to six; the net charge of the cavity; and the “hard” versus “soft” character of the ligands. These ab initio computations are used to test the reliability of the SIBFA molecular mechanics procedure, originally formulated and calibrated on the basis of ab initio computations, for such charged systems. The SIBFA intermolecular interaction energies match the corresponding ab initio values using a coreless effective potential split-valence basis set with a relative error of ≤3%. Extensions to binuclear Zn2+ complexes, such as those that occur in the Zn-binding sites of Gal4 and β-lactamase proteins, are performed to test the applicability of the methodology for such systems. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1011–1039, 2000

Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate metal ion interactions with salivary peptides histatin 3 (H3) and histatin 5 (H5). Conformational changes of these peptides in the presence of metal ions were studied using circular dichroism spectroscopy. H3 and H5 formed high affinity complexes with Cu(2+) and Ni(2+) and, to a lesser extent, with Zn(2+). Both peptides show the potential for multiple binding sites for Cu(2+) and Ni(2+) and only a single strong binding site for Zn(2+). The binding of a third Cu(2+) ion to H3 seems to enable the binding of a fourth ion to H3. The binding of a second and third Ni(2+) ion to H5 has a similar effect in enabling the binding of a fourth ion. None of the metal ions examined stabilized a regular secondary structure for either peptide. Subtle changes in overall conformation are seen with the addition of Cu(2+) to both H3 and H5.

Parallelab initio and molecular mechanics investigation of polycoordinated Zn(II) complexes with model hard and soft ligands: Variations of binding energy and of its components with number and charges of ligands

In this study we compare the binding energies of polycoordinated complexes of Zn2+ within cavities composed of model “hard” (H2O, OH−) or “soft” (CH3SH, CH3S−) ligands. Ab initio supermolecule computations are performed at the HF and MP2 levels using extended basis sets to determine the binding energies and their components as a function of: the number of ligands, ranging from three to six; the net charge of the cavity; and the “hard” versus “soft” character of the ligands. These ab initio computations are used to test the reliability of the SIBFA molecular mechanics procedure, originally formulated and calibrated on the basis of ab initio computations, for such charged systems. The SIBFA intermolecular interaction energies match the corresponding ab initio values using a coreless effective potential split-valence basis set with a relative error of ≤3%. Extensions to binuclear Zn2+ complexes, such as those that occur in the Zn-binding sites of Gal4 and β-lactamase proteins, are performed to test the applicability of the methodology for such systems. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1011–1039, 2000

The Zn-peptidase superfamily: functional convergence after evolutionary divergence

Zn-dependent carboxypeptidases (ZnCP) cleave off the C-terminal amino acid residues from proteins and peptides. Here we describe a superfamily that unites classical ZnCP with other enzymes, most of which are known (or likely) to participate in metal-dependent peptide bond cleavage, but not necessarily in polypeptide substrates. It is demonstrated that aspartoacylase (ASP gene) and succinylglutamate desuccinylase (ASTE gene) are members of the ZnCP family. The Zn-binding site along with the structural core of the protein is shown to be conserved between ZnCP and another large family of hydrolases that includes mostly aminopeptidases (ZnAP). Both families (ZnCP and ZnAP) include not only proteases but also enzymes that perform N-deacylation, and enzymes that catalyze N-desuccinylation of amino acids. This is a result of functional convergence that apparently occurred after the divergence of the two families.