The major herpes simplex virus type-1 DNA-binding protein is a zinc metalloprotein.

The primary amino acid sequence of the major herpes simplex virus type 1 (HSV-1)-infected cell polypeptide 8 (ICP8) deduced from the DNA sequence of the unique long open reading frame 29 (UL29 ORF) contains a potential metal-binding domain of the form Cys-X2-5-Cys-X2-15-A-X2-4-A where A may be either histidine or cysteine and X is any amino acid. The putative metal-binding sequence in ICP8 encompasses residues 499-512 as follows: C-N-L-C-T-F-D-T-R-H-A-C-V-H-. Atomic absorption analysis of several preparations of ICP8 indicates the presence of 1 mol of zinc/mol of protein. The zinc is resistant to removal by dialysis against concentrations of EDTA which deplete zinc from alcohol dehydrogenase. The bound zinc can be removed by reaction with the reversible sulfhydryl reagent p-hydroxymercurimethylsulfonate and the zinc-depleted protein transiently retains DNA binding activity. Digestion of both native and zinc-depleted ICP8 with V8 protease indicates that the bound zinc is required for the structural integrity of the protein.     

Structure-function analysis of the THAP zinc finger of THAP1, a large C2CH DNA-binding module linked to Rb/E2F pathways

THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of approximately 80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel beta-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.     

Expression of a RING-HC protein from rice improves resistance to Pseudomonas syringae pv. tomato DC3000 in transgenic Arabidopsis thaliana

A cDNA clone (OsRHC1) was obtained, which encodes a novel RING zinc finger protein sharing similar structural features (multiple transmembrane domains at the N-half; a unique RING zinc finger consensus Cys-X(2)-Cys-X(11)-Cys-X-His-X(3)-Cys-X(2)-Cys-X(6)-Cys-X(2)-Cys at the C terminus) to a group of closely related annotated proteins from both monocots and dicots. OsRHC1 was found to be localized on plasma membrane of rice cells and induced by wounding in rice lines containing Xa loci. Ecotopic expression of the OsRHC1 cDNA from rice (a monocot) in transgenic Arabidopsis thaliana (a dicot) enhanced the defence response toward Pseudomonas syringae pv. tomato DC3000, suggesting that OsRHC1 may confer broad-spectrum disease resistance. The protective effects were neutralized in the presence of MG132 or in an npr1-3 mutation background, indicating that the function of OsRHC1 is dependent on the ubiquitin-mediated protein degradation via the 26S proteasome and the presence of the key defence response regulator NPR1.     

Zinc release from the CH2C6 zinc finger domain of FILAMENTOUS FLOWER protein from Arabidopsis thaliana induces self-assembly

The FILAMENTOUS FLOWER gene from Arabidopsis thaliana is a member of a gene family whose role is to specify abaxial cell fate in lateral organs. Analysis of the amino-terminal region of the FILAMENTOUS FLOWER protein suggests that seven cysteine residues at positions 14, 26, 30, 33, 54, 56, and 57, and two histidine residues at positions 18 and 24 contribute to a putative zinc finger motif, Cys-X(3)-His-X(5)-His-X-Cys-X(3)-Cys-X(2)-Cys-X(20)-Cys-X-Cys-Cys. Zinc determination experiments revealed that the FILAMENTOUS FLOWER protein binds two zinc ions per molecule. Chemical modification was required to release one zinc ion, whereas the other was released spontaneously or more rapidly in the presence of metallochromic indicator. The loss of a zinc ion and the subsequent structural change of the zinc finger domain were correlated with the multimerization of the FILAMENTOUS FLOWER protein. A cysteine residue at position 56 in the FILAMENTOUS FLOWER protein potentially interferes with zinc ligation within the zinc finger and causes this zinc release. In support of this, substitution of the Cys(56) by alanine suppressed both the zinc release and the multimerization of the FILAMENTOUS FLOWER protein. Deletion analysis showed that the region between positions 45 and 107 functions in the intermolecular contacts between FILAMENTOUS FLOWER proteins. This region corresponds to the carboxyl-terminal half of the zinc finger domain and the following hydrophobic region containing two putative alpha-helices. Our results suggest that the FILAMENTOUS FLOWER protein forms a range of different conformers. This attribute may lead to a greater degree of functional flexibility that is central to its role as an abaxial cell fate regulator.     

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.     

The THAP domain: a novel protein motif with similarity to the DNA-binding domain of P element transposase

We have identified a novel evolutionarily conserved protein motif - designated the THAP domain - that defines a new family of cellular factors. We have found that the THAP domain presents striking similarities with the site-specific DNA-binding domain (DBD) of Drosophila P element transposase, including a similar size, N-terminal location, and conservation of the residues that define the THAP motif, such as the C2CH signature (Cys-Xaa(2-4)-Cys-Xaa(35-50)-Cys-Xaa(2)-His). Our results suggest that the THAP domain is a novel example of a DBD that is shared between cellular proteins and transposases from mobile genomic parasites.     

Zinc, a structural component of adenylate kinases from gram-positive bacteria.

The recent finding that Bacillus stearothermophilus adenylate kinase contains a zinc atom coordinated to four cysteines prompted us to investigate the metal-binding properties of the enzyme from various bacteria. We conclude that zinc was present only in adenylate kinase from gram-positive species and that this property is correlated with the presence of three or four Cys residues in the sequence Cys-X2-Cys-X16-Cys-X2-Cys/Asp, in which X stands for different amino acid residues.     

An enhancer trap line identifies the Drosophila homolog of the L37a ribosomal protein

A gene identified from an enhancer trap screen is shown to encode the Drosophila melanogaster homolog of the L37a ribosomal protein. The predicted 92 amino-acid sequence of this protein is 78% identical to mammalian L37a proteins, and contains a conserved Cys-X2 Cys-X14-Cys-X2-Cys zinc finger motif that may be involved in interactions with ribosomal RNA. The Drosophila L37a homolog is a single copy gene comprised of four exons and is ubiquitously expressed throughout the animal. Cytological localization reveals that Drosophila L37a maps to position 25C1-3, very near the previously described Minute mutation M(2)25C.     

Molecular basis of Bcl-xL's target recognition versatility revealed by the structure of Bcl-xL in complex with the BH3 domain of Beclin-1

Beclin-1, originally identified as a Bcl-2 binding protein, is an evolutionarily conserved protein required for autophagy. The direct interaction between Beclin-1 and Bcl-2 or Bcl-xL provides a potential convergence point for apoptosis and autophagy, two programmed cell death processes. Given the functional significance of the interaction between Beclin-1 and Bcl-2/Bcl-xL, we performed detailed biochemical and structural characterizations of this interaction. We demonstrated that the Bcl-xL-binding domain of Beclin-1 contains a BH3 domain. Therefore, Beclin-1 is a new member of the BH3-only family proteins. The structure of Bcl-xL in complex with the Beclin-1 BH3 domain was determined at high resolution by NMR spectroscopy. Although similar to other known BH3 domains, the Beclin-1 BH3 domain displays its own distinct features in the complex with Bcl-xL. Systematic analysis of all known Bcl-xL/BH3 domain complexes helped us to identify the molecular basis underlying the capacity of Bcl-xL to recognize diverse target sequences.     

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

Requirement for a zinc motif for template recognition by the bacteriophage T7 primase.

Gene 4 of bacteriophage T7 encodes two proteins, a 63 kDa and a colinear 56 kDa protein. The coding sequence of the 56 kDa protein begins at the residues encoding an internal methionine located 64 amino acids from the N-terminus of the 63 kDa protein. The 56 kDa gene 4 protein is a helicase and the 63 kDa gene 4 protein is a helicase and a primase. The unique 7 kDa N-terminus of the 63 kDa gene 4 protein is essential for primer synthesis and contains sequences with homology to a Cys4 metal binding motif, Cys-X2-Cys-X17-Cys-X2-Cys. The zinc content of the 63 kDa gene 4 protein is 1.1 g-atom/mol protein, while the zinc content of the 56 kDa gene 4 protein is < 0.01, as determined by atomic absorption spectrometry. A bacteriophage deleted for gene 4, T7 delta 4-1, is incapable of growing on Escherichia coli strains that contain plasmids expressing gene 4 proteins with single amino acid substitutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(-7)). Primase containing a substitution of the third Cys for Ser has been overexpressed in E. coli and purified to homogeneity. This mutant primase cannot catalyze template-directed synthesis of oligoribonucleotides although it is able to catalyze the synthesis of random diribonucleotides in a template-independent fashion. The mutant primase has reduced helicase activity although it catalyzes single-stranded DNA-dependent hydrolysis of dTTP at rates comparable with wild type primase. The zinc content of the mutant primase is 0.5 g-atom/mol protein.