Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts

Previous studies have shown that in a cell-free system, metallothionein (MT) releases zinc when the environment becomes oxidized and the released zinc is transferred to a zinc-binding protein if such a protein is present. However, it is unknown whether and how zinc transfers from MT to other proteins in vivo. The present study was undertaken to test the hypothesis that if zinc transfer from MT to other proteins occurs in vivo, the transfer would proceed through a direct interaction between MT and a specific group of proteins. The heart extract obtained from MT-null mice was incubated with 65Zn-MT or 65ZnCl2 and the proteins receiving 65Zn were separated by blue-native PAGE (BN-PAGE) or sodium dodecyl sulfate-PAGE (SDS-PAGE), and detected by autoradiography. A unique 65Zn-binding band was observed from the 65Zn-MT-incubated, but not the 65ZnCl2-incubated preparation. The analysis using matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry revealed that mitochondrial aconitase (m-aconitase) was among the proteins accepting Zn directly from Zn-MT. The m-aconitase, not the cytosolic aconitase (c-aconitase), was co-immunoprecipitated with MT. This study demonstrates that MT transfers zinc to m-aconitase through a direct interaction.     

Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin.

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using 65Zn indicated an association constant of 9-15 microM, with one zinc-binding site/toxin molecule. The zinc-binding site was localized to the toxin light chain as determined by binding of 65Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to Immobilon membranes. Copper was an efficient inhibitor of 65Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal-catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.     

Ions binding to S100 proteins. I. Calcium- and zinc-binding properties of bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein: Zn2+ regulates Ca2+ binding on S100b protein

Flow dialysis measurements of calcium binding to bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) proteins in 20 mM Tris-HCl buffer at pH 7.5 and 8.3 revealed that S100 proteins bind specifically 4 Ca2+ eq/mol of protein dimer. The specific calcium-binding sites had, therefore, been assigned to typical amino acid sequences on the alpha and beta subunit. The protein affinity for calcium is much lower in the presence of magnesium and potassium. Potassium strongly antagonizes calcium binding on two calcium-binding sites responsible for most of the Ca2+-induced conformational changes on S100 proteins (probably site II alpha and site II beta). Zinc-binding studies in the absence of divalent cations revealed eight zinc-binding sites/mol of S100b protein dimer that we assumed to correspond to 4 zinc-binding sites/beta subunit. Zinc binding to S100b studied with UV spectroscopy methods showed that the occupation of the four higher affinity sites and the four lower affinity sites on the protein dimer were responsible for different conformational changes in S100b structure. Zinc binding on the higher affinity sites regulates calcium binding to S100b by increasing the protein affinity for calcium and decreasing the antagonistic effect of potassium on calcium binding. Zinc-binding studies on S100a and S100 alpha alpha protein showed that the Trp-containing S100 proteins bind zinc more weakly than S100b protein. Calcium-binding studies on zinc-bound S100a proved that calcium- and zinc-binding sites were distinct although there was no increase in zinc-bound S100a affinity for calcium, as in S100b protein. Finally we provide evidence that discrepancies between previously published results on the optical properties of S100b protein probably result from oxidation of the sulfhydryl groups in the protein.     

Enhanced fluorescence resonance energy transfer between spectral variants of green fluorescent protein through zinc-site engineering

Although spectral variants of GFP should in theory be suited for fluorescence resonance energy transfer (FRET) and therefore suited for studies of protein-protein interactions, the unfavorable location of the fluorophore 15 A deep inside the GFP molecule has especially impaired this application. Here, metal-ion site engineering around the dimerization interface known from the X-ray structure of GFP is applied to the cyan and the yellow spectral variant of GFP to stabilize the heterodimeric form of these molecules and thereby increase FRET signaling. The FRET signal, determined as the ratio between the maximal emission for the yellow variant, 530 nm, and the cyan variant, 475 nm, during excitation of the cyan variant at 433 nm was increased up to 8-10-fold in the presence of 10(-4) M ZnCl2 by engineering of two symmetric metal-ion sites being either bidentate or tridentate. A similar increase in FRET signaling was however obtained in a pair of molecules in which a single bidentate metal-ion site was generated by introducing a zinc-binding residue in each of the two spectral variants of GFP and therefore creating an obligate heterodimeric pair. It is concluded that FRET signaling between spectral variants of GFP can be increased by stabilizing dimer formation and especially by favoring heterodimer formation in this case performed by metal-ion site engineering.     

Calcitermin, a novel antimicrobial peptide isolated from human airway secretions

The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide 'calcitermin' since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl(2). Analysis revealed a putative zinc-binding consensus sequence as well as an alpha-helical conformation in structure-promoting solvents.     

Matrilysin: Expression, purification, and characterization

The expression vector pGEX-2T under the control of the IPTG-inducibletac promotor is effective for the production of a fusion protein of glutathione transferase (GST, 26 kDa) and promatrilysin (28 kDa) separated from the C-terminus of GST by a thrombin cleavage site. Zwittergen (palmityl sulfobetaine), 2%, solubilizes the fusion protein that is found associated with inclusion bodies. The solubilized fusion protein is purified by affinity chromatography on GSH agarose. Promatrilysin is obtained by thrombin cleavage either on the column or after GSH elution of the fusion protein. Mono S chromatography of the recovered protein yields homogeneous promatrilysin. The zinc content of promatrilysin and its activated enzyme product is slightly greater than 2 mol of zinc per mole of protein. The results indicate that the matrix metalloproteinases (MMPs) contain two metal-binding sites at which zinc is firmly bound and possibly a third site at which it is weakly bound. Primary sequence alignments for all the MMPs have a sequence homologous to the zinc-binding site of astacin,HExxHxxGxxH, suggesting one of the zinc sites is a catalytic one, in agreement with the known inhibition of these enzymes by chelators. However, the other zinc-binding site(s) likely reflect the different ways that astacin and the MMP subfamilies are stabilized, i.e., disulfides in astacin and metal ions in the MMPs.     

Intrinsic zinc ion is essential for proper conformation of active Escherichia coli RNA polymerase

The DNA-dependent RNA polymerase (RPase) from Escherichia coli contained 2 mol of Zn/mol of holoenzyme (alpha 2 beta beta' sigma). An in vitro protocol involving sequential denaturation of RPase in 8 M urea and low pH (2.2), in the presence of 10 mM ethylenediaminetetraacetic acid (EDTA), was developed to completely remove the two intrinsic Zn ions. Subsequent reconstitution of the denatured, Zn-free RPase in the absence and presence of 10(-5) approximately 10(-4) M ZnCl2 yielded respectively the inactive apoenzyme and active (50 +/- 10%) RPase containing one Zn ion (rec-Zn1-RPase). Active rec-Cd1-RPase was similarly obtained when CdCl2 instead of ZnCl2 was used in the reconstitution. The use of 65Zn as a tracer in the two-step reconstitution procedure showed that the metal was incorporated into renatured enzyme only in the last step of reconstitution. The subunit location of the incorporated metal was identified to be in the beta subunit by the use of Affi-Gel Blue column chromatography of rec-Cd1-RPase. The analysis of apo- and rec-Zn1-RPases by sucrose density gradient sedimentation showed that the inactive apo-RPase appeared to be consisted of randomly folded protein species with S20,w values ranging from 5 to 18 S, while rec-Zn1-RPase contained a major, active 13S RPase species and a minor, inactive 7.9S species that could be separated by DNA-cellulose column chromatography. Both 13S and 7.9S RPase contained 1 mol of Zn and the five subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Zinc-dependent conformational changes in domain D5 of high molecular mass kininogen modulate contact activation.

Human high molecular mass kininogen (HK) participates as nonenzymatic cofactor in the contact system. Here, we show that recombinant domain D5 of HK (rD5) prolongs the clotting time of the intrinsic pathway of coagulation and attenuates the generation of bradykinin. Further studies indicate that a correct fold of domain D5 within HK is required for the activation of the contact system. The folding of rD5 seems to be modulated by the metal ions Zn2+, Ni2+, and Cu2+ as a specific antibody directed against the zinc-binding site in HK binds to HK and rD5 in a metal ion concentration dependent manner. The finding that these three metal ions specifically affect contact activation suggests that they regulate the accessibility of rD5 for negatively charged surfaces. Support for the assumption that the observed phenomena are due to conformational changes was obtained by fluorescence spectroscopy of rD5, demonstrating that its fluorescence spectrum was changed in the presence of ZnCl2. Moreover, negative staining electron microscopy experiments suggest that the zinc-induced changes in D5 also affect the conformation of the entire HK protein. The present data emphasize the role of zinc and other metal ions in the regulation of contact activation.     

Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses.

Human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) were purified by sucrose density gradient centrifugation in the presence of 1 mM EDTA. Pelleted gradient fractions were analyzed for total protein, total Gag capsid protein, and total zinc. Zinc was found to copurify and concentrate with the virus particles. Through successive cycles of resuspending in buffer containing EDTA and repelleting, the zinc content remained constant at about 1.7 mol of zinc per mol of Gag protein. Proteins from purified virus (HIV-1 and HTLV-I) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to polyvinylidene fluoride paper, and probed with 65ZnCl2. Viral nucleocapsid (NC) proteins (HIV-1 p7NC and HTLV-I p15NC) bound 65Zn2+. Other retroviruses, including simian immunodeficiency virus, equine infectious anemia virus, bovine leukemia virus, Moloney murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus, were found to contain amounts of zinc per milligram of total protein similar to those found in HIV-1 and HTLV-I. Collectively, these data support the hypothesis that retroviral NC proteins function as zinc finger proteins in mature viruses.     

Identification of zinc-binding sites of proteins: zinc binds to the amino-terminal region of tubulin

The discovery that certain proteins may require zinc for their activity, and the fact that several of them cannot be purified in large amounts, has led us to develop a rapid, sensitive method to detect these proteins in samples. This method is based on the fractionation of the proteins by gel electrophoresis, blotting onto nitrocellulose paper, and overlaying with 65Zn. We have tested the procedure with well-characterized zinc-binding proteins. In the case of tubulin, we have used this method to localize its zinc-binding site. It was found that zinc binds to the first 150 amino acids of both alpha- and beta-tubulin subunits.     

Formation, circular dichroism and x-ray photoelectron spectroscopy of hepatic Zn-thionein.

The formation of the powerful Zn binding protein called Zn-thionein was examined using male albino rats and [14C]cysteine, as cystein is known to be the most abundant constituent of this metal protein. 65% of the hepatic [14C]cysteine was incorporated into the protein portion of freshly prepared Zn-thionein. The protein was isolated by a combination of ethanol/chloroform treatment and various chromatographic steps, including ion exchange and gel filtration. 4.7 mol of Zn, 0.02 mol of Cd and less than 0.001 mol of either Cu or Hg were found per 12 000 g of portein. It was presumed that considerable amounts of Zn were lost during these isolation procedures, with the consequence of disulphide gridge formation. Indeed, the presence of R-S-S-R was deduced from circular dichroism and X-ray photoelectron spectroscopy. Due to the clearly detectable disulphide chromophore in the circular dichroism spectrum, it was possible to assign the shoulder at S 2p1/2,3/2 = 162.7 eV of the X-ray photoelectron spectrum of native Zn-thionein to R-S-S-R and not to strongly polarized sulphur. Upon reducing R-S-S-R-containing native Zn-thionein with dithiothreitol, all oxidised thiolate moieties of the thionein molecule could be restored. The addition of ZnCl2 with the subsequent desalting of extraneously bound Zn2 yielded a homogeneous Zn-thionein with 9.6 mol Zn2 per mol protein. A stoichiometry of ZnRS 1:3 was seen, which confirmed earlier reports of the existence of the mixed Cd,Zn-thionein. The conversion of mixed Cd,Zn-thionein into homogeneous Zn-, Cd-, Hg- and Cu-thionein by the gel filtration technique proved successful. From chiroptical measurements, the extraordinary contribution of the metal chromophores to the circular dichroism was seen. Due to the differences in the geometry of complexes formed by the respective metal ions, dramatic changes in the protein portion were expected. Polyacrylamide disc electrophoresis of purified native untreated Zn-thionein resulted in the appearance of two or more bands. This phenomenon was attributed to the different migration rates of cystine-thionein and thiolate-rich Zn-thionein, and was consistent with the spectral properties of the above Zn-protein species. By contrast, only one single band was monitored when a homogeneous metal-thionein was electrophoresed.     

棉花秸秆催化热解特性及动力学的研究

通过热重分析实验观察K2CO3、KOH、KCl、NaCl、MgCl2和ZnCl26种无机催化剂对棉花秸秆热解催化效果的影响。K2CO,、KOH处理过的棉花秆与纯棉花秆相比热解发生在较低的温度范围,而KCl、NaCl、MgCl,和ZnCl2处理过的棉花秆热解发生在较高的温度范围。碱性催化剂K2C03、KOH降低了棉花秸秆的最大质量损失率,而KCl、NaCl、MgCl2和ZnCl2却增大了棉花秸秆的最大质量损失率。应用Ozawa动力学模型得到动力学参数,棉花秸秆在热解主要阶段可由一段一级反应过程描述,升温速率10K／min时活化能值EA的范围是35～66kJ／mol,频率因子自然对数的范围是4～12。     

The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein

The recent demonstration that the NS5A protein of hepatitis C virus (HCV) contains an unconventional zinc-binding site with the format Cx(17)CxCx(20)C and the presence of a similar sequence element in the NS5A proteins of members of the Pestivirus genus has led to the hypothesis that the NS5A protein of the pestivirus bovine viral diarrhea virus (BVDV) is a zinc-binding protein. A method for the expression and partial purification of BVDV NS5A was developed, and the partially purified protein was analyzed for zinc content by atomic absorption spectroscopy. BVDV NS5A was found to coordinate a single zinc atom per protein molecule. Mutation of any of the four cysteines of the predicted zinc-binding motif eliminated zinc coordination. Furthermore, analysis of mutations at these cysteine residues in the context of a BVDV replicon system indicated that these residues were absolutely essential for RNA replication. The recently determined crystal structure of the N-terminal zinc-binding domain of the HCV NS5A protein, combined with secondary structure predictions of the region surrounding the mapped BVDV zinc-binding region, indicates that the BVDV zinc-binding motif fits the general template Cx(22)CxCx(24)C and likely comprises a three-stranded antiparallel beta-sheet fold. These data highlight the similarities between the Hepacivirus and Pestivirus NS5A proteins and suggest that both proteins perform a not-yet-defined function in RNA replication that requires coordination of a single zinc atom.     

A catalytically active fragment of cGMP-dependent protein kinase. Occupation of its cGMP-binding sites does not affect its phosphotransferase activity

Treatment of cGMP-dependent protein kinase with low concentrations of trypsin generates an enzyme fragment of 65 kDa which is fully active in the absence of cGMP. The fragment has a s20,w value of 4.6 S indicating that the active fragment is a monomer of 65 kDa. Trypsin removes the first 77 amino acids which contain the aminoterminal dimerization site and the autophosphorylation sites. The Km and Vmax values of the fragment for ATP and Kemptide were essentially the same as those for the native enzyme. The fragment binds 2 mol cGMP/mol fragment with affinities close to that of the native enzyme. However, binding of cGMP to these sites was non-cooperative and shows similar characteristics to the autophosphorylated native enzyme. These results indicate that the aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.     

Characterization of the low mol. wt zinc-binding ligand from rat small intestine by comparison to the organic zinc-binding ligands

1. 65Zn complexes of picolinate (PA), citrate (CA), L-histidine (L-his), arachidonic acid (AA) or low mol. wt zinc-binding ligand from rat intestine (LMW-ZBL) gave 65Zn eluting peak fraction numbers of 53, 53, 56, 59 and 59 respectively, in a Sephadex G-75 column chromatography. 2. The 65Zn eluting peak fraction numbers with CA, L-his, PA, prostaglandin (PG)E2, AA, no ligand, arachidonate (AT) or LMW-ZBL were 49, 50, 54, 55, 58, 64, 75 and 76 respectively in a Sephadex G-25 column chromatography. 3. In a Sephadex G-15 column chromatography, the 65Zn eluting peak fraction numbers with CA, PGE2, AA, L-his, LMW-ZBL or PA were 49, 50, 51, 52, 52 and 55 respectively. 4. The LMW-ZBL in rat small intestine appears to be an AA-like substance.     

Amino acid sequence and characterization of a protein inhibitor of protein kinase C

The complete primary structure has been determined for an inhibitor protein of protein kinase C. The bovine brain-derived inhibitor has a pI of 6 and its N-terminal alanine residue is blocked by acetylation. Fragments obtained by chemical and enzymatic cleavage of the purified inhibitor were analyzed by Edman degradation, fast atom bombardment mass spectrometry, and tandem mass spectrometry. The results establish that the protein has a calculated average molecular mass of 13,690 daltons and contains 125 amino acid residues with the following sequence: (sequence: see text) The inhibitor does not show significant homology with any other known protein. Circular dichroism of the freshly prepared apoprotein indicated a secondary structural content of 23% alpha-helix, 31% beta-sheet, and 11% beta-turn. Immobilization on nitrocellulose followed by exposure to a 65Zn2(+)-containing overlay solution showed that, like protein kinase C itself, the inhibitor is a zinc-binding protein, although the sequence does not reveal a "zinc finger" structure. Competition with 10-fold molar excess Ca2+ or Mg2+ did not reduce the zinc-binding specificity of this inhibitor.     

Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity

Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity. LF bound ⁶⁵Zn both in solution and on protein blots. The ⁶⁵Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.     

A Zinc Site in the C-Terminal Domain of RAG1 Is Essential for DNA Cleavage Activity

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn²⁺ ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn²⁺ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn²⁺ ions. Stripping the full complement of bound Zn²⁺ ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn²⁺ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn²⁺ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn²⁺ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn²⁺ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn²⁺ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.     

Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.