title: Alternative splicing of Wilms' tumor suppressor protein modulates DNA binding activity through isoform-specific DNA-induced conformational changes.
abstract: The Wilms' tumor suppressor protein (WT1) is a zinc finger transcription factor that appears to function differently according to the presence of a posttranscriptional modification that adds three amino acids into one of the linker regions between the zinc fingers. We have investigated the structural consequences of the insertion of the Lys-Thr-Ser (KTS) sequence by preparing recombinant protein constructs of the four zinc finger DNA-binding domain of WT1 corresponding to the two isoforms with (+KTS) and without (-KTS) the insertion, which is located in the linker region between the third and fourth zinc fingers. NMR resonance assignments were used to estimate the structural differences between the two isoforms both free in solution and in complex with a 14 base pair DNA duplex corresponding to the WT1 recognition element. The NMR spectra indicate that the two isoforms are nearly identical in structure in the absence of the DNA. Only the immediate region of the insertion showed any change in chemical shifts. Upon DNA binding, the NMR spectrum of each isoform changed to indicate greater structure formation in the linker regions. Significant differences were observed between the spectra of the DNA complexes of the +KTS and -KTS isoforms, with the -KTS construct forming a more stable complex, consistent with prior biochemical assays. The majority of the differences between the spectra of the two complexes occur in the immediate region of the insertion, which appears to be closer in structure to the free form of the protein in the case of the +KTS complex. The insertion of the KTS sequence disrupts important interactions of the linker region with the adjacent zinc fingers, thus lowering the stability of the complex. The "normal" (-KTS) sequence of the linker appears to be involved in a C-terminal helix-capping interaction with the helix of the preceding zinc finger, a stabilizing interaction which is abrogated in the +KTS isoform.
authors.affiliation: Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10596 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Laity
authors.forename: J
authors.initials: J
authors.lastname: Chung
authors.forename: H J
authors.initials: HJ
authors.lastname: Dyson
authors.forename: P E
authors.initials: PE
authors.lastname: Wright
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D051766:Early Growth Response Protein 1; D016162:Genes, Wilms Tumor / Q000235:genetics*; D006801:Humans; D006860:Hydrogen Bonding; D017874:Immediate-Early Proteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D020033:Protein Isoforms; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013489:Suppression, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D025721:WT1 Proteins; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014157:Transcription Factors; D025721:WT1 Proteins; D004247:DNA