• New Vistas in Zinc Finger Biochemistry: Examining the Metal-Mediated DNA Recognition of the Neural Zinc Finger Factor/Myelin Transcription Factor Family of Non-Classical Zinc Finger Proteins and Creating Catalytic Moieties from Zinc Finger Scaffolds

      Besold, Angelique Nicole; Michel, Sarah L. J. (2014)
      The Neural Zinc Finger Factor/Myelin Transcription Factor (NZF/MyT) family of `non-classical' zinc fingers (ZFs) is involved in regulating key genes during neuronal development. These proteins contain domains with a CCHHC motif and utilize a CCHC ligand set to coordinate zinc ions in a tetrahedral geometry and adopt secondary structure. To better understand the metal-mediated DNA recognition properties of two members of this family, NZF-1 and MyT1, we have taken a biochemical/biophysical approach involving UV-visible spectroscopy, circular dichroism, and fluorescence anisotropy. Our first efforts focused on the role of metal ion identity in function and utilized a two domain construct of NZF-1 comprised of ZFs two and three (NZF-1-F2F3), which bind an AAGTT DNA sequence located in the promoter of the β-retinoic acid receptor gene (βRAR). We discovered that iron(II) can coordinate to the ZF domains with no alteration in the DNA binding properties when compared to the zinc bound form. We then utilized a mutagenesis approach to determine the role of a conserved histidine that is not involved in metal coordination. We determined that this residue is involved in stabilizing a hydrogen bonding interaction important for NZF-1-F2F3/βRAR binding. Furthermore, we demonstrated that a single arginine residue in ZF3 of NZF-1, which is absent in MyT1, is required for the NZF-1-F2F3/βRAR DNA interaction. These results suggest that the few non-conserved amino acids present in the ZF domains of this family drive sequence-specific DNA recognition. In addition, research aimed at engineering ZF domains to have catalytic activity has been pursued. In one effort, a prototype classical CCHH ZF domain called Consensus Peptide-1 (CP-1) was mutated at one of the metal coordinating cysteines. This resulted in an open coordination sphere at the zinc site, making it accessible for hydrolysis of substrates. Using 4-nitrophenylacetate as a probe, we demonstrated that this construct can promote hydrolysis. We also created a chimeric protein composed of the RNA binding ZF tristetraprolin (TTP) and the RNA cleavage protein Ribonuclease4 (RNase4). This Chimera, TTP2D-RNase4, catalytically cleaves target RNA in vitro. This is the first step towards our long term goal of engineering TTP as a novel anti-inflammatory/anti-cancer agent.