Journal of Molecular Biology
Volume 372, Issue 5, 5 October 2007, Pages 1227-1245
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Structure of the Wilms Tumor Suppressor Protein Zinc Finger Domain Bound to DNA

https://doi.org/10.1016/j.jmb.2007.07.017Get rights and content

Abstract

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys2His2 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.

Introduction

Wilms tumor (WT) or nephroblastoma is a pediatric kidney cancer that was first described by Max Wilms in 1899.1 It is one of the most frequently occurring solid tumors of childhood, affecting about 1 in 10,000 children, typically between ages two and four years.1 WT serves as a paradigm for understanding the relationship between loss of developmental control and gain of tumorigenic potential.2 In particular, loss of function of tumor suppressor genes has been implicated in the development of WT; the Wilms tumor suppressor gene WT1 on chromosome 11p13 was the second tumor suppressor gene to be cloned after the retinoblastoma gene RB-1.

The Wilms tumor suppressor gene encodes a DNA-binding protein containing four Cys2His2 zinc fingers.3., 4. The Cys2His2 zinc finger (zf) motif is employed by a diverse array of transcription factors that play an important role in cellular signal transduction. Over 1000 copies of this motif have been identified in vertebrate genomes, making it one of the predominant mediators of sequence-specific protein–DNA interactions. In addition to its C-terminal DNA-binding domain, the Wilms tumor protein also contains a proline/glutamine-rich N terminus, activation and repression domains, nuclear localization signals, and at least two self-association domains.5., 6., 7., 8.

The cellular expression pattern of WT1 is tissue-specific and also depends on the growth stage of the organism. WT1 is essential for normal urogenital development, and mutations in the WT1 gene have been associated with diseases, such as Denys-Drash,9 WAGR,10 and Frasier11 syndromes. A combination of alternative splicing, alternative translation start sites, and RNA editing leads to the expression of at least 24 different isoforms that share four C-terminal zinc fingers and an N-terminal proline/glutamine-rich regulatory region.12., 13. Of particular interest are two alternative splice sites at the end of exon 9 that lead to insertion of three amino acids (KTS) after the glycine in the canonical TGEKP linker sequence between zinc fingers 3 and 4.13 This alternative splice site is highly conserved during evolution and is found in all vertebrates. The relative abundance of these splice forms is constant; developmental abnormalities are associated with altered ratios of +KTS and −KTS isoforms.13., 14., 15. Mounting evidence indicates that +KTS and −KTS isoforms perform distinct biological functions and differ in their nucleic acid binding properties. The −KTS isoform binds DNA sequence-specifically and appears to function primarily in transcriptional regulation; more than 30 putative target genes have been identified, the majority of which contain the EGR-1 consensus site in their promoters.1., 16. The more abundant +KTS splice variant binds RNA, associates preferentially with components of the pre-mRNA splicing machinery and appears to function in post-transcriptional regulatory processes.12., 13., 17., 18. NMR chemical shift mapping19 and relaxation experiments20 show that insertion of the KTS sequence modulates DNA binding through isoform-specific DNA-induced conformational changes, and leads to increased linker flexibility and loss of DNA binding by zf4, hence providing a molecular basis for the differential affinity of +KTS and −KTS variants for DNA.20

The amino acid sequences of the Wilms tumor zinc fingers are homologous to those of other well-characterized zinc finger domains (Figure 1(a)). The Cys and His ligands are invariant, and the core hydrophobic residues and linker motifs are all highly conserved. The linkers perform a “snap-lock” helix-capping function to stabilize the complexes of these multi-finger proteins with DNA.21 Fingers 2–4 of WT1 are closest in sequence (65% identity) to fingers 1–3 of zif268 (EGR-1); although finger 1 of WT1 contains the canonical zinc finger sequence motif (Ar-X-C-X2-4-C-X3-Ar-X5-L-X2-H-X3-4-H), it has a lower homology with the other WT1 or Zif268 fingers in the characteristic base recognition motif at the tip of the finger (Figure 1(a)).

Initial experiments to identify WT1 binding sites showed that the protein preferentially binds to DNA sequences related to the EGR-1 consensus binding site 5′-GCG-(T/G)GG-GCG-3′.22., 23. Subsequent binding site selection studies have demonstrated that the highest affinity DNA binding site for WT1 has a consensus sequence of 5′-GCG-(T/G)GG-GAG-(T/G)(T/G/A)(T/G)-3′.23., 24., 25., 26., 27. The high affinity binding sequence has been identified in the promoters of two WT1-responsive genes.16 Sequence-specific DNA recognition is mediated by fingers 2–4. Finger 1 plays only a secondary role in DNA binding; it contributes only modestly to binding affinity and specificity and does not significantly protect the DNA in footprinting experiments.24., 25., 27., 28. Deletion of finger 1 results in only a twofold to fivefold decrease in binding affinity, depending on the nucleic acid sequence.27 A systematic analysis of mutant DNA consensus sequences indicates specific recognition of most base-pairs, with the wild-type sequences showing the highest affinity for WT1–KTS.26

In order to elucidate the structural basis for differences in DNA binding between the three-finger Zif268 and the four-finger WT1 proteins and to obtain insights into the structural basis by which Denys-Drash and other disease-associated mutations affect DNA binding, we determined the structure of the C-terminal zinc finger domain of the WT1–KTS splice form bound to 14 bp and 17 bp DNA oligonucleotides containing the consensus EGR-1 binding site (Figure 1(b)), the binding site found in the majority of target gene promoters. The shorter 14 bp oligonucleotide was previously used in NMR dynamics studies.19 The structure of the complex of zf1-4 with the 14 bp DNA duplex obtained using X-ray crystallography shows that zinc fingers 2–4 make base-specific contacts in the major groove of the DNA duplex, while zf1 interacts only with the phosphodiester DNA backbone and with a flipped out cytidine base at a frayed end of the double helix. Examination of the structure in solution using NMR methods show that zf1 lies in the major groove of both the 14 bp and a longer 17 bp oligonucleotide. However, this finger appears to interact less extensively with the DNA than zf2-4 and makes no base-specific contacts. The structures provide new insights into sequence-specific recognition of DNA by WT1 and provide a framework for understanding the structural and mechanistic effects of disease-causing mutations.

Section snippets

Crystal structure of zf1-4 with 14 bp DNA duplex

The WT1 zf1-4 (−KTS) protein (residues 318–435, with an N-terminal alanine and excluding the alternative splice sequence KTS between residues 407 and 408) and the 14 bp DNA oligonucleotide representing the EGR-1 consensus site, was originally designed for NMR studies.19 However, during the acquisition and analysis of NMR spectra, crystals formed in the NMR tube upon storage in the refrigerator. These crystals were suitable for X-ray diffraction analysis and were indexed in hexagonal space group

Sequence requirements in the consensus DNA

The overall structure of fingers 2 to 4 of WT1–KTS bound to the 14 bp DNA duplex closely resembles the conformation found for fingers 1 to 3 of zif268.29., 37., 38. Three consecutive fingers bind DNA by wrapping around the major groove and make contact with 10 bp. Except for the linker following finger 1, the conserved threonine and glycine residues of the other linkers in the WT1–KTS–DNA complex form backbone and side-chain hydrogen bonds that are exactly analogous to those formed by the

Expression, purification, and complex formation

The carboxy-terminal zinc finger domain of the Wilms tumor suppressor protein (WT1) was expressed and purified as published.19., 20. This domain encompasses residues 318–435 of the WT1 protein, without the insertion of the Lys-Thr-Ser sequence between residues 407 and 408 (WT1 + KTS). Therefore, this domain is referred to as the −KTS form of the WT1 protein. In addition, this construct contains an N-terminal Ala due to the cloning procedure.20 Uniformly 15N- and 15N/13C-isotope enriched samples

Acknowledgements

We are grateful to Linda Tennant for help with sample preparation, to John Chung and Gerard Kroon for expert technical advice regarding the set up of NMR experiments, and to Andreas Heine and Xiaoping Dai for advice with the X-ray experiments. This work was supported by the National Institutes of Health grant GM36643 and by the Skaggs Institute for Chemical Biology. R.S. gratefully recognizes support from the Skaggs Institute for Chemical Biology, the Deutscher Akademischer Austauschdienst

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    Present addresses: R. Stoll, Ruhr-University of Bochum, Faculty of Chemistry and Biochemistry, D-44801 Bochum, Germany; B. M. Lee, Department of Chemistry and Biochemistry, Southern Illinois University, 224 Neckers Hall, Carbondale, IL 62901, USA; E. W. Debler, Laboratory of Cell Biology, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA; J. H. Laity, Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2400, USA.

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