Solution structure of the FCS zinc finger domain of the human polycomb group protein L(3)mbt‐like 2

Polycomb group proteins are epigenetic regulators that maintain patterns of gene expression over multiple rounds of cell division. Many of these proteins, including polyhomeotic and the MBT repeat containing proteins SCM and dSfmbt, contain an atypical C2C2 zinc finger with a characteristic phenylalanine–cysteine–serine sequence motif. The reoccurrence of this so‐called FCS zinc finger in a variety of polycomb group proteins suggests that it has an important regulatory function. We have determined the solution structure of the FCS zinc finger of the human dSfmbt homologue L(3)mbt‐like 2 (L3MBTL2). The structure consists of a β‐hairpin followed by an α‐helix. The zinc ligands are situated in the β‐hairpin and at the N‐terminus of the α‐helix an arrangement typical of the treble clef class of zinc fingers. The structure is consistent with the proposal that FCS zinc fingers bind to regulatory RNAs.     

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)     

Identification of a gene in Leishmania infantum encoding a protein that contains a SP-RING/MIZ zinc finger domain

The SP-RING or Miz zinc finger domain that is related to the classical RING-finger motif, defines a class of proteins that can act as E3-like factors in the pathway of small ubiquitin-related modifier (SUMO) conjugation. This family includes the mammalian protein inhibitor of activated STAT (PIAS) proteins and related proteins from lower eukaryotes. Here we report the existence of a gene in Leishmania infantum, present as two identical copies placed upstream of each MAT2 gene copy, and transcribed as a single approximately 2.2 kb mRNA both in the logarithmic and stationary phases of the promastigote stage. This gene encodes a 47 kDa protein that has been named LORIEN. LORIEN is circumscribed to the cell periphery and it is antigenic during L. infantum infection of dogs and hamsters. Strikingly, this novel protein contains a highly conserved SP-RING/Miz zinc finger domain, raising the possibility that a SUMO or ubiquitin-like system may exist in this microorganism.     

Solution structure of the zinc finger HIT domain in protein FON

The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 A for the structured residues 12-48. The fold consists of two consecutive antiparallel beta-sheets and two short C-terminal helices packed against the second beta-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain.     

A KRAB domain zinc finger protein in imprinting and disease

The monoallelic expression of imprinted genes is regulated by DNA methylation marks that originate from the oocyte or sperm. Li et al. (2008) show in this issue of Developmental Cell that the KRAB zinc finger protein Zfp57 contributes to the embryonic maintenance of these imprints. At one locus, Zfp57 is also involved in imprint establishment. These findings provide a mechanistic interpretation for Mackay et al.'s recently reported ZFP57 mutations in patients with transient neonatal diabetes.     

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.     

The zinc finger domain of IKKγ (NEMO) protein in health and disease

Inhibitor of κB kinase (IKK) gamma (IKKγ), also known as nuclear factor κB (NF-κB) essential modulator (NEMO), is a component of the IKK complex that is essential for the activation of the NF-κB pathway. The NF-κB pathway plays a major role in the regulation of the expression of genes that are involved in immune response, inflammation, cell adhesion, cell survival and development. As part of the IKK complex, IKKγ plays a regulatory role by linking the complex to upstream signalling molecules. IKKγ contains two coiled-coil regions, a leucine zipper domain and a highly conserved zinc finger domain. Mutations affecting IKKγ have been associated with X-linked hypohidrotic ectodermal dysplasia with immune deficiency (HED-ID), with the majority of these mutations affecting the C-terminal region of the protein where the zinc finger is located. The zinc finger of IKKγ is needed for NF-κB activation in a cell- and stimulus-specific manner. The major mechanism by which the zinc finger plays this role appears to be the recognition of polyubiquitinated upstream signalling intermediates. This assertion reinforces the current notion that ubiquitination plays a major role in mediating protein–protein interactions in the NF-κB signalling pathway. Because the zinc finger domain of IKKγ is very likely involved in mediating interactions with ubiquitinated proteins, investigations that look for upstream activators or inhibitors of the IKK complex that bind to and interact with the zinc finger of IKKγ are required to gain a better insight into the exact roles of this domain and into the pathogenesis of HED-ID.     

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.     

Structure of the ubiquitin-binding zinc finger domain of human DNA Y-polymerase eta

The ubiquitin-binding zinc finger (UBZ) domain of human DNA Y-family polymerase (pol) eta is important in the recruitment of the polymerase to the stalled replication machinery in translesion synthesis. Here, we report the solution structure of the pol eta UBZ domain and its interaction with ubiquitin. We show that the UBZ domain adopts a classical C(2)H(2) zinc-finger structure characterized by a betabetaalpha fold. Nuclear magnetic resonance titration maps the binding interfaces between UBZ and ubiquitin to the alpha-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single alpha-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol eta UBZ domain represents a novel member of the C(2)H(2) zinc finger family that interacts with ubiquitin to regulate translesion synthesis.