Cooperative binding of the yeast Spt10p activator to the histone upstream activating sequences is mediated through an N-terminal dimerization domain

The yeast Spt10p activator is a putative histone acetyltransferase (HAT) possessing a sequence-specific DNA-binding domain (DBD) which binds to the upstream activation sequences (UAS elements) in the histone gene promoters. Spt10p binds to a pair of histone UAS elements with extreme positive cooperativity. The molecular basis of this cooperativity was addressed. Spt10p (640 residues) is an elongated dimer, but the isolated DBD (residues 283–396) is a monomer and binds non-cooperatively to DNA. A Spt10p fragment comprising the N-terminal domain (NTD), HAT domain and DBD (residues 1–396) binds cooperatively and is a dimer, whereas an overlapping Spt10p fragment comprising the DBD and C-terminal domains (residues 283–640) binds non-cooperatively and is a monomer. These observations imply that cooperative binding requires dimerization. The isolated NTD (residues 1–98) is a dimer and is responsible for dimerization. We propose that cooperativity involves a conformational change in the Spt10p dimer which facilitates the simultaneous recognition of two UAS elements. In vivo, deletion of the NTD results in poor growth, but does not prevent the binding at the HTA1 promoter, suggesting that dimerization is biologically important. Residues 1–396 are sufficient for normal growth, indicating that the critical functions of Spt10p reside in the N-terminal domains.     

Structural analysis of a mutant of the HIV-1 integrase zinc finger domain that forms a single conformation

HIV-1 integrase consists of three functional domains, an N-terminal zinc finger domain, a catalytic core domain and a C-terminal DNA binding domain. NMR analysis of an isolated N-terminal domain (IN(1-55)) has shown that IN(1-55) exists in two conformational states [E and D forms; Cai et al. (1997) Nat. Struct. Biol. 4, 567-577]. The two forms differ in the coordination of the zinc ion by two histidine residues. In the present study, structural analysis of a mutant of IN(1-55), Y15A, by NMR spectroscopy indicated that the mutant protein folds correctly but takes only the E form. Since the Y15A mutation abrogates the HIV-1 infectivity, Y15 might have some important role in the full-length integrase activity during the virus infection cycle. Our results suggest a possible role of Y15 in structural transition between the E and D forms of HIV-1 integrase to allow the optimal tetramerization.     

Functional analyses of the Dof domain, a zinc finger DNA-binding domain, in a pumpkin DNA-binding protein AOBP

AOBP, a DNA-binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA-binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA-binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA-binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.     

C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)     

The yeast putative transcriptional repressor RGM1 is a proline-rich zinc finger protein

I have cloned a yeast gene, RGM1, which encodes a proline-rich zinc, finger protein. rgm1 mutants do not show any obvious phenotype but overexpression of RGM1 gene greatly impairs cell growth. The proline-rich region of RGM1 attached to a heterologous DNA binding domain is able to repress the expression of the target gene. RGM1 shares similar zinc finger motifs with the mammalian Egr (early growth response) proteins as well as proline-rich sequences with a high serine and threonine content, suggesting that RGM1 and Egr proteins could have functional similarities.     

The finger domain of simian virus 40 large T antigen controls DNA-binding specificity.

The specificity and regulation of protein-DNA interactions play a crucial role in all aspects of communication between genotype and phenotype in a cell. The large T antigen of simian virus 40 binds to identical, yet quite differently arranged, pentanucleotide motifs in the simian virus 40 control region, sites I and II. Wild-type T antigen preferentially binds site I. We demonstrate that a bacterial peptide encoding residues 1 to 259 (T260) includes the essential amino acids required for binding to both DNA sites but predominantly binds site II. However, a longer peptide (residues 1 to 369; T370) binds almost exclusively to site I. Thus, the addition of amino acids 260 to 369 to the T260 peptide results in the loss of site II binding. This region includes a single putative metal-binding region, and mutation of T370 at either conserved cysteine of the finger results in equal but inefficient binding to both sites. While no metal binding has been shown to be directly associated with this sequence, these results suggest a novel, perhaps structural, function for such a finger motif, since this domain of T antigen appears to play a crucial role in modulating the DNA-binding behavior of T-antigen peptides.     

The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function

Clp ATPases are unique chaperones that promote protein unfolding and subsequent degradation by proteases. The mechanism by which this occurs is poorly understood. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) involved in substrate recognition. ZBD forms a very stable dimer that is essential for promoting the degradation of some typical ClpXP substrates such as lambdaO and MuA but not GFP-SsrA. Furthermore, experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Removal of ZBD from the ClpX sequence renders the ATPase activity of ClpX largely insensitive to the presence of ClpP, substrates, or the SspB cofactor. All these results indicate that ZBD plays an important role in the ClpX mechanism of function and that ATP binding and/or hydrolysis drives a conformational change in ClpX involving ZBD.     

A factor that regulates the class II major histocompatibility complex gene DPA is a member of a subfamily of zinc finger proteins that includes a Drosophila developmental control protein.

A novel DNA sequence element termed the J element involved in the regulated expression of class II major histocompatibility complex genes was recently described. To study this element and its role in class II gene regulation further, a cDNA library was screened with oligonucleotide probes containing both the S element and the nearby J element of the human DPA gene. Several DNA clones were obtained by this procedure, one of which, clone 18, is reported and characterized here. It encodes a protein predicted to contain 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box) here. One of the Drosophila sequences, ttk, is a developmental control gene, while a second does not contain a zinc finger region but encodes a structure important in oocyte development. An acidic activation domain is located between the N-terminal conserved region of clone 18 and its zinc fingers. This protein appears to require both the S and J elements, which are separated by 10 bp for optimal binding. Antisense cDNA to clone 18 inhibited the expression of a reporter construct containing the DPA promoter, indicating its functional importance in the expression of this class II gene.     

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.