The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity

The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.     

Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase.

Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His-X2-His. This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging. In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition. We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity. Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate. The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme. The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry. The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity.     

Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.     

The zinc-dependent redox switch domain of the chaperone Hsp33 has a novel fold

The Escherichia coli chaperone Hsp33 contains a C-terminal zinc-binding domain that modulates activity by a so-called "redox switch". The oxidized form in the absence of zinc is active, while the reduced form in the presence of zinc is inactive. X-ray crystal structures of Hsp33 invariably omit details of the C-terminal domain, which is truncated in protein constructs that are capable of forming crystals. We report the solution structure of a recombinant 61-residue protein containing the zinc-binding domain (residues 227-287) of Hsp33, in the presence of stoichiometric amounts of Zn2+. The zinc-bound protein is well folded, and forms a novel structure unlike other published zinc-binding domains. The structure consists of two helices at right-angles to each other, a two-stranded B-hairpin and a third helix at the C terminus. The zinc site comprises the side-chains of the conserved cysteine residues 232, 234, 262 and 265, and connects a short sequence before the first helix with the tight turn in the middle of the B-hairpin. The structure of the C-terminal zinc-binding domain suggests a mechanism for the operation of the redox switch: loss of the bound zinc ion disrupts the folded structure, allowing the ligand cysteine residues to be oxidized, probably to disulfide bonds. The observation that the C-terminal domain is poorly structured in the active oxidized form suggests that the loss of zinc and unfolding of the domain precedes the oxidation of the thiolate groups of the cysteine residues, since the formation of disulfides between distant parts of the domain sequence would presumably promote the formation of stable three-dimensional structure in the oxidized form.Hsp33 provides an example of a redox signaling system that utilizes protein folding and unfolding together with chemical modification for transduction of external stimuli, in this case oxidative stress, to activate the machinery of the cell that is designed to deal with that stress.     

Activation and induction of glycine N-methyltransferase by retinoids are tissue- and gender-specific

Betaine-homocysteine S-methyltransferase (BHMT; EC2.1.1.5) is a zinc metalloenzyme that catalyzes the transfer of a methyl group from betaine to homocysteine to produce dimethylglycine and Met, respectively. This enzyme is a member of a family of zinc-dependent methyltransferases that use thiols or selenols as methyl acceptors and which contain the following motif: G[ILV]NCX(20, 100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I. We recently reported that the three cysteine residues within this motif function as ligands to zinc in BHMT because changing any of them to alanine abolished zinc-binding and enzyme activity (A. P. Breksa, III, and T. A. Garrow, 1999, Biochemistry 38, 13991-13998). To determine if other amino acid residues in this motif were critical for enzyme function, the two regions defined by the motif in human BHMT, GVNCH(218) and VRYIGGCCGFEPYHI(307), were subjected to semirandom and random site-directed mutagenesis. Mutant enzymes were classified as either active or inactive based on their ability to complement the Met auxotrophy of Escherichia coli strain J5-3. The Gly residue at position 214 was found to be absolutely essential for complementation. The positions occupied by Gly297, Gly298, and Gly301 favored substitutions of small amino acids like Ala and Ser. We hypothesize that these Gly residues provide the necessary flexibility to the Zn-binding region to permit coordination of the metal.     

Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.

The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (< 5%). Site-directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non-chelating residue, alanine, abolished not only the metal-binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc-binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.     

Genetic analysis of a potential zinc-binding domain of the adenovirus E4 34k protein

E4 34k, the product of adenovirus early region 4 (E4) open reading frame 6, modulates viral late gene expression, viral DNA replication, apoptosis, double strand break repair, and transformation through multiple interactions with components in infected and transformed cells. Conservation of several cysteine and histidine residues among E4 34k sequences from a variety of adenovirus serotypes suggests the presence of a zinc binding domain important for function. Consistent with the hypothesis that E4 34k is a zinc metalloprotein, zinc binding by baculovirus-expressed E4 34k protein was demonstrated in a zinc blotting assay. To investigate the relationship between the potential zinc-binding region and E4 34k function, a series of mutant genes containing single amino acid substitutions at each of the conserved cysteine and histidine residues in E4 34k were constructed. The mutant proteins were examined for the ability to complement the late protein synthetic defect of an E4 deletion mutant, to physically interact with the viral E1b 55-kDa protein (E1b 55k) and cellular p53 protein, to relocalize E1b 55k, and to destabilize the p53 protein. These analyses identified a subset of cysteine and histidine residues required for stimulation of late gene expression, physical interaction with E1b 55k, and p53 destabilization. These data suggest that a zinc-binding domain participates in the formation of the E4 34k-E1b 55k physical complex and that the complex is required in late gene expression and for p53 destabilization.     

Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif. Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis.

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme. Glu(129) may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.     

A zinc-binding protein in a metal-resistant strain, Alcaligenes eutrophus CH34.

Synthesis of a zinc-binding protein was induced when Alcaligenes eutrophus CH34 was grown in the presence of high concentrations of zinc (2.3 mM). The zinc-binding protein has a low content of cysteine and a high content of acidic amino acids and, thus, differs from metallothionein.     

Zinc is required for assembly and function of the anti-trp RNA-binding attenuation protein,AT

The anti-TRAP protein (AT) of Bacillus subtilis regulates expression of the trp operon and other genes concerned with tryptophan metabolism. AT acts by inhibiting the tryptophan-activated trp RNA-binding attenuation protein (TRAP). AT is an oligomer of identical 53-residue polypeptides; it is produced in response to the accumulation of uncharged tRNA(Trp). Each AT polypeptide has two cysteine-rich clusters that correspond to the signature motif of the cysteine-rich zinc-binding domain of the chaperone protein DnaJ. Here we characterize the putative zinc-binding domain of AT and establish the importance of zinc for AT assembly and activity. AT is shown to contain Zn(II) at a ratio of one ion per monomer. Bound zinc is necessary for maintenance of the quaternary structure of AT; the removal of zinc converts the AT complex into inactive monomers. All four cysteine residues in the AT polypeptide are involved in Zn(II) coordination. Chemical cross-linking analyses indicate that the AT functional oligomer is a hexamer composed of two trimers. Substituting alanine for any cysteine residue of AT results in rapid degradation of the mutant protein in vivo. We propose a model for the AT trimer in which three AT chains are held together by three zinc atoms, each coordinated by the N-terminal segment and the C-terminal segment of separate AT polypeptides.     

Evidence from extended X-ray absorption fine structure and site-specific mutagenesis for zinc fingers in UvrA protein of Escherichia coli

The UvrA protein is the damage recognition subunit of the Escherichia coli repair enzyme ABC excision nuclease. Sequence analysis of this 940-amino acid protein revealed two regions of sequence homology to the zinc finger motif found in many DNA binding proteins. Physical and chemical analyses indicate about 2 zinc atoms/molecule. We have used extended x-ray absorption fine structure analysis to demonstrate that each of these zinc atoms is coordinated with 4 cysteine residues at a distance of 2.32 +/- 0.2 A. Substitution of one of the cysteines by a histidine, a serine, or an alanine in one of the potential finger sites resulted in a respective decrease in complementing activity. We thus conclude that the two zinc fingers identified by sequence analysis do indeed have zinc finger structure in UvrA protein.     

Zinc-binding subunits of yeast RNA polymerases

The zinc-binding subunits of yeast RNA polymerase A(I) and B(II) have been identified by a zinc-blotting technique. The two largest subunits of each enzyme (A190, A135, B220, and B150), as well as A12.2, A10, B44.5, B12.6, and B10, bind 65Zn(II). Predicted zinc-binding motifs have been noted in the NH2-terminal part of B220 and the COOH-terminal region of B150 subunits. Subdomains encompassing these motifs have been overproduced as MalE-fusion proteins and shown to retain zinc binding activity. Site-directed mutagenesis in the predicted metal-binding domain of B150 demonstrated its role in zinc binding. Mutations of cysteine residues C1163, C1166, C1182, and C1185 affected 65Zn2+ binding in vitro and caused a lethal or thermosensitive phenotype for growth. The ability to bind zinc is not sufficient for function since mutations in vicinal residues not affecting zinc binding were either lethal or thermosensitive. The role of zinc in RNA polymerase structure and function is discussed in the light of the present results.     

Cytidine deaminases from B. subtilis and E. coli: compensating effects of changing zinc coordination and quaternary structure.

Cytidine deaminase from E. coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits (M(r) = 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy. Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine. A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E. coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B. H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468]. This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E. coli (His-102, Cys-129, and Cys-132). Instead, the B. subtilis and E. coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence alignments, together with model building, suggest one possible mechanism of compensation.     

Hyperthermophilic topoisomerase I from Thermotoga maritima. A very efficient enzyme that functions independently of zinc binding

Topoisomerases, by controlling DNA supercoiling state, are key enzymes for adaptation to high temperatures in thermophilic organisms. We focus here on the topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima (optimal growth temperature, 80 degrees C). To determine the properties of the enzyme compared with those of its mesophilic homologs, we overexpressed T. maritima topoisomerase I in Escherichia coli and purified it to near homogeneity. We show that T. maritima topoisomerase I exhibits a very high DNA relaxing activity. Mapping of the cleavage sites on a variety of single-stranded oligonucleotides indicates a strong preference for a cytosine at position -4 of the cleavage, a property shared by E. coli topoisomerase I and archaeal reverse gyrases. As expected, the mutation of the putative active site Tyr 288 to Phe led to a totally inactive protein. To investigate the role of the unique zinc motif (Cys-X-Cys-X(16)-Cys-X-Cys) present in T. maritima topoisomerase I, experiments have been performed with the protein mutated on the tetracysteine motif. Strikingly, the results show that zinc binding is not required for DNA relaxation activity, contrary to the E. coli enzyme. Furthermore, neither thermostability nor cleavage specificity is altered in this mutant. This finding opens the question of the role of the zinc-binding motif in T. maritima topoisomerase I and suggests that this hyperthermophilic topoisomerase possesses a different mechanism from its mesophilic homolog.     

Mutations in residues involved in zinc binding in the catalytic site of Escherichia coli threonyl-tRNA synthetase confer a dominant lethal phenotype

Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNA(Thr) isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNA(Thr) by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.     

The Finger Domain of the Human Deubiquitinating Enzyme HAUSP is a Zinc Ribbon

Human deubiquitinating enzyme HAUSP is a cysteine protease that regulates the levels of the tumor suppressor protein p53. By comparative sequence and structural analysis, we show that the previously uncharacterized finger domain insert to the catalytic core of HAUSP is a zinc ribbon that has lost its zinc-binding ability.     

Zinc stoichiometry of yeast RNA polymerase II and characterization of mutations in the zinc-binding domain of the largest subunit

Atomic absorption spectroscopy demonstrated that highly purified RNA polymerase II from the yeast Saccharomyces cerevisiae binds seven zinc ions. This number agrees with the number of potential zinc-binding sites among the 12 different subunits of the enzyme and with our observation that the ninth largest subunit alone is able to bind two zinc ions. The zinc-binding motif in the largest subunit of the enzyme was investigated using mutagenic analysis. Altering any one of the six conserved residues in the zinc-binding motif conferred either a lethal or conditional phenotype, and zinc blot analysis indicated that mutant forms of the domain had a 2-fold reduction in zinc affinity. Mutations in the zinc-binding domain reduced RNA polymerase II activity in cell-free extracts, even though protein blot analysis indicated that the mutant subunit was present in excess of wild-type levels. Purification of one mutant RNA polymerase revealed a subunit profile that was wild-type like with the exception of two subunits not required for core enzyme activity (Rpb4p and Rpb7p), which were missing. Core activity of the mutant enzyme was reduced 20-fold. We conclude that mutations in the zinc-binding domain can reduce core activity without altering the association of any of the subunits required for this activity.     

Structural origins of amino acid selection without editing by cysteinyl-tRNA synthetase

Cysteinyl-tRNA synthetase (CysRS) is highly specific for synthesis of cysteinyl adenylate, yet does not possess the amino acid editing activity characteristic of many other tRNA synthetases. To elucidate how CysRS is able to distinguish cysteine from non-cognate amino acids, crystal structures of the Escherichia coli enzyme were determined in apo and cysteine-bound states. The structures reveal that the substrate cysteine thiolate forms a single direct interaction with a zinc ion bound at the base of the active site cleft, in a trigonal bipyramidal geometry together with four highly conserved protein side chains. Cysteine binding induces movement of the zinc ion towards substrate, as well as flipping of the conserved Trp205 indole ring to pack on the thiol side chain. The imidazole groups of five conserved histidines lie adjacent to the zinc ion, forming a unique arrangement suggestive of functional significance. Thus, amino acid discrimination without editing arises most directly from the favorable zinc-thiolate interaction, which is not possible for non-cognate substrates. Additional selectivity may be generated during the induced-fit conformational changes that help assemble the active site.