Kinetic behaviour of WT 1's zinc finger domain in binding to the alpha-actinin-1 mRNA.

The zinc finger transcription factor Wilms tumour protein (WT 1) is known for its essential involvement in the development of the genitourinary system as well as of other organs and tissues. WT 1 is capable of selectively binding either DNA or mRNA targets. A KTS insertion due to alternative splicing between the zinc fingers 3 and 4 and an unconventional zinc finger 1 are the unique features that distinguish WT 1 from classical DNA-binding C(2)H(2)-type zinc finger proteins. The DNA binding characteristics of WT 1 are well studied. Due to lack of information about its native RNA targets, no extensive research has been directed at how WT 1 binds RNA. Using surface plasmon resonance, this study attempts to understand the binding behaviour of WT 1 zinc fingers with its recently reported and first putative mRNA target, ACT 34, whose stem-loop structure is believed to be critical for the interactions with WT 1. We have analysed the interactions of five WT 1 zinc finger truncations with wild-type ACT 34 and four variants. Our results indicate that WT 1 zinc fingers bind ACT 34 in a specific manner, and that this occurs as interplay of all four zinc fingers. We also report that a sensitive kinetic balance, which is equilibrated by both zinc finger 1 and KTS, regulates the interaction with ACT 34. The stem-loop and the flanking nucleotides are important elements for specific recognition by WT 1 zinc fingers.

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.

Porphyrin binding and distortion and substrate specificity in the ferrochelatase reaction: the role of active site residues.

The specific insertion of a divalent metal ion into tetrapyrrole macrocycles is catalyzed by a group of enzymes called chelatases. Distortion of the tetrapyrrole has been proposed to be an important component of the mechanism of metallation. We present the structures of two different inhibitor complexes: (1) N-methylmesoporphyrin (N-MeMP) with the His183Ala variant of Bacillus subtilis ferrochelatase; (2) the wild-type form of the same enzyme with deuteroporphyrin IX 2,4-disulfonic acid dihydrochloride (dSDP). Analysis of the structures showed that only one N-MeMP isomer out of the eight possible was bound to the protein and it was different from the isomer that was earlier found to bind to the wild-type enzyme. A comparison of the distortion of this porphyrin with other porphyrin complexes of ferrochelatase and a catalytic antibody with ferrochelatase activity using normal-coordinate structural decomposition reveals that certain types of distortion are predominant in all these complexes. On the other hand, dSDP, which binds closer to the protein surface compared to N-MeMP, does not undergo any distortion upon binding to the protein, underscoring that the position of the porphyrin within the active site pocket is crucial for generating the distortion required for metal insertion. In addition, in contrast to the wild-type enzyme, Cu(2+)-soaking of the His183Ala variant complex did not show any traces of porphyrin metallation. Collectively, these results provide new insights into the role of the active site residues of ferrochelatase in controlling stereospecificity, distortion and metallation.