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.     

Soluble expression and purification of tumor suppressor WT1 and its zinc finger domain

Full length murine WT1 and its zinc finger domain were separately inserted into Escherichia coli expression vectors with various fusion tags on either terminus by Gateway technology (Invitrogen) and expression of soluble protein was assessed. Fusion proteins including the four zinc finger domains of WT1 were used to optimize expression and purification conditions and to characterize WT1:DNA interactions in the absence of WT1:WT1 interactions. Zinc finger protein for in vitro characterization was prepared by IMAC purification of WT1 residues 321-443 with a thioredoxin-hexahistidine N-terminal fusion, followed by 3C protease cleavage to liberate the zinc fingers and cation exchange chromatography to isolate the zinc fingers and reduce the level of the truncated forms. Titration of zinc finger domain with a binding site from the PDGFA promoter gave a K(d) of 100±30nM for the -KTS isoform and 130±40nM for the +KTS isoform. The zinc finger domain was also co-crystallized with a double-stranded DNA oligonucleotide, yielding crystals that diffract to 5.5?. Using protocols established for the zinc finger domain, we expressed soluble full-length WT1 with an N-terminal thioredoxin domain and purified the fusion protein by IMAC. In electro-mobility shift assays, purified full-length WT1 bound double-stranded oligonucleotides containing known WT1 binding sites, but not control oligonucleotides. Two molecules of WT1 bind an oligonucleotide presenting the full PDGFA promoter, demonstrating that active full-length WT1 can be produced in E. coli and used to investigate WT1 dimerization in complex with DNA in vitro.     

Recognition mechanism of Wilms’ tumour suppressor protein and DNA triplets: insights from molecular dynamics simulation and free energy analysis

The Wilms’ tumour suppressor protein (WT1) plays a multifaceted role in human cancer processes. Mutations on its DNA recognition domain could lead to Denys–Drash syndrome, and alternate splicing results in insertion of the tripeptide Lys–Thr–Ser (KTS) between the third and fourth zinc fingers (ZFs), leading to changes in the DNA-binding function. However, detailed recognition mechanisms of the WT1–DNA complex have not been explored. To clarify the mutational effects upon WT1 towards DNA binding at the atomic level, molecular dynamics simulations and the molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) method were employed. The simulation results indicate that mutations in ZF domains (E427Q and Q369H) may weaken the binding affinity, and the statistical analyses of the hydrogen bonds and hydrophobic interactions show that eight residues (Lys351, Arg366, Arg375, Arg376, Lys399, Arg403, Arg424 and Arg430) have a significant influence on recognition and binding to DNA. Insertion of the tripeptide KTS could form an immobilized hydrogen-bonding network with Arg403, affecting the flexibility and angle of the linker between ZF3 and ZF4, thus influencing the recognition between the protein and the DNA triplet at its 5′ terminus. These results represent the first step towards a thorough characterization of the WT1 recognition mechanisms, providing a better understanding of the structure–function relationship of WT1 and its mutants.     

Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affinities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-higher affinity for the WTE sequence compared with that of the EGR-1 binding motif. Mutational analysis of the WTE motif revealed a significant contribution to binding affinity by the adenine nucleotide at the eighth position (5'GCGTGGGAGT3') as well as by the 3'-most thymine (5'GCGTGGGAGT3'), whereas mutations in either flanking nucleotides or other nucleotides in the core sequence did not significantly affect the specific binding affinity. Mutations within WT1 zinc fingers II to IV abolished the sequence-specific binding of WT1 to WTE, whereas alterations within the first WT1 zinc finger reduced the binding affinity approximately 10-fold but did not abolish sequence recognition. We have thus identified a WT1 target, which, although similar in sequence to the EGR-1 motif, shows a 20- to 30-fold-higher affinity for WT1. These results suggest that physiological action of WT1 is mediated by binding sites of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is discussed in the context of recent structural analysis.     

The C-terminal zinc finger of UvrA does not bind DNA directly but regulates damage-specific DNA binding

In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding.     

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.     

Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition

The connection of functional modules is effective for the design of DNA binding molecules with the desired sequence specificity. C(2)H(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules and are expected to recognize any DNA sequences by permutation, multi-connection, and the substitution of various sets of zinc fingers. To investigate the effects of the replacement of the terminal finger on the DNA recognition by other fingers, we have constructed the three zinc finger peptides with finger substitution at the N- or C-terminus, Sp1(zf223), Sp1(zf323), and Sp1(zf321). From the results of gel mobility shift assays, each mutant peptide binds preferentially to the target sequence that is predicted if the fingers act in a modular fashion. The methylation interference analyses demonstrate that in the cases of the N-terminal finger substitution mutants, Sp1(zf223) and Sp1(zf323), the N-terminal finger recognizes bases to different extents from that of the wild-type peptide, Sp1(zf123). Of special interest is the fact that the N-terminal finger of the C-terminal finger substitution mutant, Sp1(zf321), shows a distinct base recognition from those of Sp1(zf123) and Sp1(zf323). DNase I footprinting analyses indicate that the C-terminal finger (active finger) induces a conformational change in the DNA in the region for the binding of the N-terminal finger (passive finger). The present results strongly suggest that the extent of base recognition of the N-terminal finger is dominated by the binding of the C-terminal finger. This information provides an important clue for the creation of a zinc finger peptide with the desired specificity, which is applicable to the design of novel drugs and biological tools.