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.     

Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.     

The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA breaks and nicks and the consequent enzyme activation. Other structures recognize intact DNA

The recognition of double-stranded DNA breaks and single-stranded nicks by human poly(ADP-ribose) polymerase and the consequent enzymic activation were examined using derivatives of the enzyme expressed in Escherichia coli. The N-terminal 162 residues encompass two zinc fingers. Deletion or mutation of the first finger results in a loss of activation by DNA with either single-stranded or double-stranded damage. Destruction of the second finger reduces activation by double-stranded DNA breaks only slightly, but eliminates activation by single-stranded DNA nicks. These data suggest that activation by single-stranded DNA nicks requires two zinc fingers, but activation by double-stranded DNA breaks requires only the finger closer to the N terminus. Variant proteins that lack both zinc fingers are enzymically inactive but still exhibit weak DNA binding, which is independent of DNA damage. Thus, other regions are also capable of binding intact DNA, but the recognition of a strand nick or break which occasions the synthesis of poly(ADP-ribose) specifically requires the zinc fingers.     

Zinc fingers in sex determination: only one of the two C. elegans Tra-1 proteins binds DNA in vitro.

The tra-1 gene of Caenorhabditis elegans is a major developmental regulator that promotes female development. Two mRNAs are expressed from the tra-1 locus as a result of alternative mRNA processing. One mRNA encodes a protein with five zinc fingers and the other a protein with only the first two zinc fingers. We have derived a preferred in vitro DNA binding site for the five finger protein by selection from random oligonucleotides. The two finger protein does not bind to DNA in vitro. Moreover, removal of the first two fingers from the five finger protein does not eliminate binding and has little effect on its preferred binding site. We find that a protein sequence amino-terminal to the finger domain also appears to play a role in DNA binding.     

Expression, purification, and characterization of the 4 zinc finger region of human tumor suppressor WT1

Wilm's Tumor gene 1 (WT1) encodes a zinc finger protein with four distinct splice isoforms. WT1 has a critical role in genesis of various cancer types both at the DNA/RNA and the protein level. The zinc-finger DNA-binding capacity of the protein is located in the C-terminal domain. Two recombinant proteins, 6HIS-ZN-wt1 and 6HIS-ZN+wt1, corresponding to two alternative splice variants of the C-terminal regions of human WT1 (-KTS) and WT1 (+KTS), respectively, were over-expressed with hexa-histidine fusion tags in inclusion bodies in Escherichia coli for crystallization studies. A combination of Ni2+-NTA affinity and size-exclusion chromatography was applied for purification of the proteins in denaturing conditions. The effects of various buffers, salts and other additives were scrutinized in a systematic screening to establish the optimal conditions for solubility and refolding of the recombinant WT1 proteins. Circular dichroism analysis revealed the expected betabetaalpha content for the refolded proteins, with a notable degradation of the alpha-helical segment in the DNA-free state. Electrophoretic mobility shift assay with double-stranded DNA containing the double Egr1 consensus site 5'-GCG-TGG-GCG-3' confirmed that 6HIS-ZN-wt1 has higher DNA binding affinity than 6HIS-ZN+wt1.     

GATA-1 bends DNA in a site-independent fashion

The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.     

Kin17, a mouse nuclear zinc finger protein that binds preferentially to curved DNA.

Kin17 is a 45 kDa protein encoded by the KIN17 gene located on mouse chromosome 2, band A. The kin17 amino acid sequence predicts two domains, which were shown to be functional: (i) a bipartite nuclear localization signal (NLS) that can drive the protein to the cell nucleus, (ii) a bona fide zinc finger of the C2H2 type. The zinc finger is involved in kin17 binding to double-stranded DNA since a mutant deleted of the zinc finger, kin17 delta 1, showed reduced binding. Single-stranded DNA was bound poorly by kin17. Interestingly, we found that kin17 protein showed preferential binding to curved DNA from either pBR322 or synthetic oligonucleotides. Binding of kin17 to a non-curved DNA segment increased after we had inserted into it a short curved synthetic oligonucleotide. Kin17 delta 2, a mutant deleted of 110 amino acids at the C-terminal end, still exhibited preferential binding to curved DNA and so did kin17 delta 1, suggesting that a domain recognizing curved DNA is located in the protein core.     

Pentaprobe: a comprehensive sequence for the one-step detection of DNA-binding activities

The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence-specific DNA-binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 4⁵ possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 4⁵ × 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double-stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA-binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N- and C-terminal fingers of GATA-1. We also showed that the N-terminal zinc finger domain of FOG-1 does not behave as a typical DNA-binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.     

重组PLZF蛋白锌指结构域的表达、提纯和活性分析

PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成。鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯。为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点。构建好的表达质粒转化到BL21 (DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达。用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上。对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础。