Zn-binding Motif Research Articles

The Zinc Finger of the CSN-Associated Deubiquitinating Enzyme USP15 Is Essential to Rescue the E3 Ligase Rbx1

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.

Expression of carbonic anhydrase-related protein CA-RP VIII in non-small cell lung cancer.

Carbonic anhydrase-related protein (CA-RP VIII) lacks a Zn-binding motif which is essential for carbonic anhydrase activity. Therefore, CA-RP VIII is believed to have a no catalytic activity and a new biological property. In the present study, CA-RP VIII expression in non-tumorous lung and non-small cell lung carcinomas was investigated. Little or no expression of CA-RP VIII was observed in human lungs by Northern-blot analysis. Reverse-transcription polymerase chain reaction analysis demonstrated CA-RP VIII mRNA in developing human lungs and, to a lesser extent, in normal lungs. Subsequent immunohistochemical staining revealed that CA-RP VIII was expressed in the pulmonary epithelium in developing lungs; however, CA-RP VIII expression was restricted in bronchial ciliated cells in adult lungs. Neither bronchial gland nor squamous metaplasia of interstitial pneumonia expressed CA-RP VIII. In contrast, CA-RP VIII was strongly expressed in almost all archival lung cancer specimens, which included 24 squamous cell carcinomas, 25 adenocarcinomas, and 6 adenosquamous cell carcinomas. Cancer cells at the front of tumor progression expressed CA-RP VIII in particular abundance. The present findings suggest that CA-RP VIII may play a role in non-small lung cell carcinomas.

Arg362 and Tyr365 of the Botulinum Neurotoxin Type A Light Chain Are Involved in Transition State Stabilization

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).

Aspergillus nidulans DigA, a potential homolog of Saccharomyces cerevisiae Pep3 (Vps18), is required for nuclear migration, mitochondrial morphology and polarized growth.

The fungal vacuole is an acidic organelle that is involved in a variety of physiological processes, such as protein turnover, ion and pH homeostasis and osmoregulation. The function of the vacuole largely depends on vesicle transport providing the organelle with enzymes and substrates. The process of vesicle transportation has been studied best in Saccharomyces cerevisiae, where several proteins that are crucial for intracellular vesicle sorting have been identified. One such protein is Pep3 (Vps18). In pep3 mutants vacuole function and vacuole morphology are affected. We cloned the gene for a potential homolog of Pep3 from the filamentous fungus Aspergillus nidulans. The gene, digA (for dichotomous growth), was identified in a screen for nuclear migration mutants. A. nidulans digA encodes a protein of 108.3 kDa, which includes a 122-amino acid clathrin repeat motif, two short coiled-coil regions, and a RING finger Zn-binding motif at the C-terminus. All three sequence motifs suggest interaction of DigA with other proteins. DigA is 25% identical to a homolog from Schizosaccharomyces pombe, 23% to a protein from human, 21% to the product of the Drosophila melanogaster gene deep orange (dor) and 18% to S. cerevisiae Pep3 (Vps18). We localized DigA as a HA-epitope-tagged protein in the cytoplasm of A. nidulans vegetative cells, by secondary immunofluorescence. A digA mutant displays a pleiotropic phenotype with clustered mitochondria, clustered nuclei and a defect in polarization of the actin cytoskeleton. These results suggest that vacuolar functions are required for organelle positioning and polarized growth.

Characterization of Rous sarcoma virus Gag particles assembled in vitro.

Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.

Isolation and Molecular Characterization of the COP 1 Gene Homolog from Rice, Oryza sativa L. subsp. Indica var. Pusa Basmati 1

The COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1) gene has been identified earlier from dicot species namely Arabidopsis, tomato and pea. The protein encoded by this gene acts as a molecular switch that negatively regulates the transition from the skotomorphogenic to the photomorphogenic mode of plant development. We have isolated and characterized the COP1 homolog from a monocot species, i.e. rice (var. Pusa Basmati 1). All the functional domains (Zn-binding RING finger motif, coiled-coil region, WD-40 repeats, cytoplasmic/nuclear localization sequences and protein-protein interaction domains) that are known in the COP1 proteins from dicots are conserved in COP1 from rice as well. The transcript levels of COP1 vary in various tissues of the rice plant. These variations were found to be development-dependent and do not solely depend on the light conditions.

Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.

Zn(2+) ions selectively induce antimicrobial salivary peptide histatin-5 to fuse negatively charged vesicles. Identification and characterization of a zinc-binding motif present in the functional domain.

The salivary antimicrobial peptide histatin-5 is able to aggregate and fuse negatively charged small unilamellar vesicles, and this fusogenic activity is selectively induced by the presence of zinc ions. Circular dichroism spectroscopy shows that histatin-5, in the presence of negatively charged vesicles and zinc ions, undergoes a conformational change leading to the stabilization of an alpha-helical secondary structure. We attribute the specific action of the zinc ions to the presence of a consensus sequence, HEXXH, located in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif in many proteins. Two-dimensional proton NMR spectroscopy of histatin-5 in a trifluoroethanol/water mixture (a membrane mimetic environment) has been performed and the results analyzed by means of distance geometry and restrained molecular dynamics simulations. Our results reveal that the peptide chain, including the Zn-binding consensus sequence corresponding to residues 15-19, is in a helicoidal conformation. Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide. The mechanism of action of this antimicrobial peptide is discussed on the basis of its structural characteristics with particular attention to the Zn-binding motif.

Soluble Human Endothelin-Converting Enzyme-1: Expression, Purification, and Demonstration of Pronounced pH Sensitivity

Endothelin-converting enzyme-1 (ECE-1) is a type II integral membrane protein that belongs to a family of metalloproteases which includes ECE-2, neprilysin (neutral endopeptidase 24.11, EC 3.4.24.11), and Kell blood group protein. ECE-1 cleaves its biologically inactive native substrate, big endothelin-1, to generate a powerful vasoactive 21-amino acid peptide, endothelin-1. ECE-1 consists of a short N-terminal cytoplasmic tail, a transmembrane hydrophobic domain, and a large extracellular domain containing the catalytic site with a conserved Zn-binding motif. We have constructed a secreted, soluble form of ECE-1 (solECE-1) by fusing the cleavable N-terminal signal sequence of human alkaline phosphatase in frame with the entire extracellular domain of ECE-1. Stable transfectant CHO cell lines expressing up to 6.1 mg of solECE-1 per liter culture medium were established and solECE-1 was purified to homogeneity using three chromatographic steps with a 24% yield. SolECE-1 behaves as a dimer of 110-kDa subunits. SolECE-1 has a sharp pH optimum, similar to the native form, ECE-1a, but has a slightly more acidic pH optimum of 6.1–6.4 than that of 6.7–6.9 for ECE-1a. At its optimal pH of 6.4, solECE-1 cleaved big ET-1:big ET-2:big ET-3 in a ratio of 8.1:1:1.4, was inhibited by phosphoramidon with an IC50value of 0.35 ± 0.05 μM, had aKmvalue of 4.65 ± 0.78 μM for big ET-1, and had akcatvalue of 5.82 ± 0.21 min−1, all values comparable to those for ECE-1a at its optimal pH of 6.8. Phosphoramidon inhibition of both ECE-1a and solECE-1 is highly pH-dependent. At pH 5.8, phosphoramidon inhibited ECE-1a and solECE-1 with IC50values of 14 and 33 nM, respectively, which are 49- and 1224-fold more potent than at pH 7.2. SolECE-1 is highly glycosylated, similar to ECE-1a. Deglycosylation of solECE-1 by peptide N-glycosidase F shifted the apparent molecular weight of solECE-1 to approximately 80 kDa and the deglycosylated form(s) of solECE-1 preserved at least 72% of the activity of the glycosylated form.

Mutational analysis of a satellite phage activator.

The late gene activator, Delta, of satellite phage P4 is more efficient than the Delta of satellite phage phiR73 in utilizing a P2 helper prophage that lacks an activator (ogr) gene. Analysis of P4 Delta is complicated by the fact that this protein contains two tandem phiR73 Delta-like domains. We performed a mutational analysis of phiR73 Delta, in order to select mutations that might not be found using P4 Delta. The host RNA polymerase alpha subunit mutation rpoA155 (L289F) blocks the growth of P2, P4, and P4 carrying the delta gene of phiR73. A mutant of this latter phage that can grow in the presence of rpoA155 carries a V19M mutation in phiR73 Delta. This suggests that aa 19 contacts RNA polymerase, in addition to the aa residues 13, 42 and 44, that have been implicated in interactions with RNA polymerase by previous mutational analyses of P2 ogr and P4 delta. In corroboration of the proposed role of the regions at aa residues 19, 42, and 44, we found phiR73 Delta mutations in these regions that showed a reduced activation of late gene expression, but a normal ability to bind to late gene promoters. All activators in the Delta class contain four Cys residues that bind Zn2+. Mutation of these aa residues in phiR73 Delta eliminated late gene activation. Spectroscopic analysis of these mutant proteins revealed that they were unable to bind Zn2+. Histidine residues were substituted for two of the Cys residues (C30 and C35), changing a C2C2 type Zn-binding motif to a C2H2 motif. Although His residues are used in coordinating Zn2+ in other proteins, these His substitutions resulted in complete loss of activity and the inability to bind Zn2+.

Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions.

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

Mutational Analyses Support a Model for the HRV2 2A Proteinase

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity. A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase. The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular (“cis”) and intermolecular (“trans”) activities. The HRV2 proteinase 2A is proposed to have a two domain structure, with the catalytic site and substrate binding region on one face of the molecule and a Zn-binding motif on the opposite face. Residues Gly 123, Gly 124, Thr 121, and Cys 101 are proposed to be involved in the architecture of the substrate binding pocket and to provide the correct environment for the catalytic triad of His 18, Asp 35, and Cys 106. Residues Tyr 85 and Tyr 86 are thought to participate in substrate recognition. The presence of an extensive C-terminal helix, in which Asp 132, Arg 134, Phe 130, and Phe 136 play important roles, explains why mutations in this region are generally detrimental to proteinase activity. The proposed Zn-binding motif comprises Cys 52, Cys 54, Cys 112, and His 114. Exchange of these residues inactivates the enzyme. Furthermore, as measured by atom emission spectroscopy, Zn was absent from purified preparations of proteinase 2A in which His 114 had been replaced by Asn. The absence of disulphide bridges was confirmed by subjecting highly purified HRV2 proteinase 2A to one- and two-step alkylation procedures.

Identification of the gene coding for the largest subunit of RNA polymerase I (A) of Drosophila melanogaster.

The gene coding for the largest subunit (RPA1) of RNA polymerase I (A) of Drosophila melanogaster (DmRPA1) was cloned and sequenced. The gene is interrupted by seven small introns and the cDNA reveals an open reading frame of 4932 nucleotides. The deduced polypeptide consists of 1644 amino acids with a calculated molecular weight of 185 kDa. Although the protein sequence exhibits the specific pattern of conserved regions found in all RNA polymerase largest subunits characterized so far, the overall sequence similarity among the RPA1 subunits of different species is much lower than seen with the corresponding subunits of RNA polymerases II and III. Two highly divergent hydrophilic domains characteristic for RPA1 separate the conserved blocks a and b in the N-terminal region and blocks g and h in the C-terminal section, respectively. In both cases the distance between the homologous blocks is enlarged by about 70 amino acids relative to the largest subunits of RNA polymerases II and III, and the corresponding subunit of the archaebacterial enzyme. Compared with RPA1 sequences of lower eukaryotes, the C-terminal hydrophilic domain in DmRPA1 is similar in length and acidity whereas the N-terminal domain is slightly shorter but retains the same basicity. The sequence insertions do not feature common motifs, suggesting a role for them in the interaction of RNA polymerase I with proteins required for the species-specific transcription of rDNA. The RPA1 subunits of Drosophila melanogaster and lower eukaryotes share an additional Zn-binding motif at the N-terminus with archaebacterial and RPC1 subunits, testifying to the complex evolutionary relationships among the RNA polymerases.

Genomic Organization of the Zinc-Endopeptidase Astacin

The crayfish digestive protease astacin is the first described member of the astacin family of zinc-endopeptidases, for which it is regarded as a prototype. We have isolated and characterized the genomic sequence of astacin which spans a region of 2616 bp. The coding sequence is distributed over five exons and is interrupted by four introns. It was observed that structurally and functionally essential units of the protein, like the three α-helices, the five β-strands, the Zn-binding motif, and the Met turn are never disrupted by introns. The start site of transcription was determined by primer extension analysis, confirming the existence of a pre-pro-protein of 49 amino acids which so far had not been detectable at the protein level. In addition, when compared to the amino acid sequence of mature astacin, a carboxy-terminal extension of two additional amino acids was also found. The exon–intron pattern of the astacin gene was compared to those of three other astacin family members with known genomic sequences, i.e., tolloid ofDrosophila,the fish hatching enzyme LCE, and the human BMP1 gene. In each of the four proteins one intron was found to be inserted in the codon for a similar Gly residue which is highly conserved in this position within the astacin family.

Spectroscopic characterization of rhinoviral protease 2A: Zn is essential for the structural integrity.

Recently, protease 2A of human rhinovirus 2 (HRV2 2A) was shown to require a zinc ion for the formation of an active enzyme although zinc is not involved mechanistically. The data presented clearly show that the zinc ion bound to a picornaviral-specific motif represents an essential component of the native structure, probably representing a new Zn-binding motif. This structure, containing mostly beta-strand elements as shown by CD spectroscopy, changes drastically upon removal of zinc. The zinc-depleted form does represent an intermediate with mostly unchanged secondary structure, but not a fully denatured random coil as obtained by guanidinium hydrochloride. This is indicated by the blue-shifted fluorescence spectra and by CD. The native protein exhibited a cooperative phase transition at 53 degrees C. In contrast, the zinc-depleted form did not show any transition at all, again demonstrating the stabilizing role of the zinc ion. A structural intermediate was observed during thermal and pH denaturation that may represent a molten globule, as suggested by its ANS binding.