New insights into DNA-binding behavior of Wilms tumor protein (WT1)--a dual study.
Wilms Tumor suppressor protein (WT1) is a transcription factor that is involved in a variety of developmental functions during organ development. It is also implicated in the pathology of several different cancer forms. The protein contains four C(2)H(2)-type zinc fingers and it specifically binds GC-rich sequences in the promoter regions of its target genes, which are either up or down regulated. Two properties make WT1 a more unusual transcription factor - an unconventional amino acid composition for zinc finger 1, and the insertion of a tri-peptide KTS in some of the splice isoforms of WT1. Using six WT1 constructs in which zinc fingers are systematically deleted, a dual study based on a bacterial 1-hybrid system and surface plasmon resonance measurements is performed. The experiments show that the effect of zinc finger 1 is not significant in terms of overall DNA-binding kinetics, however it influences both the specificity of target recognition and stability of interaction in presence of KTS. The KTS insertion, however, only mildly retards binding affinity, mainly by affecting the on-rate. We suggest that the insertion disturbs zinc finger 4 from its binding frame, thus weakening the rate of target recognition. Finally, for the construct in which both zinc fingers 1 and 4 were deleted, the two middle fingers 2-3 still could function as a 'minimal DNA-recognition domain' for WT1, however the formation of a stable protein-DNA complex is impaired since the overall affinity was dramatically reduced mainly since the off-rate was severely affected.
Center for Molecular Protein Science, Lund University, Getingevägen 60, 221 00, Lund, Sweden.
Elmar
E
Nurmemmedov
Raymond K
RK
Yengo
Hüseyin
H
Uysal
Robert
R
Karlsson
Marjolein M G M
MM
Thunnissen
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D017353:Gene Deletion; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D025721:WT1 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D025721:WT1 Proteins; D004247:DNA