CCHX Zinc Finger Derivatives Retain the Ability to Bind Zn(II) and Mediate Protein-DNA Interactions

Abstract

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a ␤␤␣ fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krü ppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krü ppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.

ThensdCGene Encoding a Putative C2H2-Type Transcription Factor Is a Key Activator of Sexual Development inAspergillus nidulans

“…The C 2 HC motif is found in various eukaryotic proteins that have diverse functions such as chromatin acetylation (Akhtar and Becker 2001;Smith et al 2005), DNA repair ( Jones et al 1988), and development control (Raabe et al 2004;Xie et al 2005). Many C 2 HC motifs bind to DNA (Lee et al 1998;Simpson et al 2003) or interact with motifs of other transcription factors (Sadowski et al 2003). Some motifs can combine with C 2 H 2 -or C 2 C 2 GATA-type motifs (McLaughlin et al 1994;Haenlin et al 1997;Hahm et al 1998).…”

Structural and Biophysical Analysis of the DNA Binding Properties of Myelin Transcription Factor 1

“…For each titration, 25-30 injections of 5-10 l of titrant were made at 5-min intervals. Data were corrected for heats of dilution from control experiments of DNA into buffer, and analyzed using Origin ITC Analysis software (MicroCal Software, Northampton, MA), as described previously (17).…”

Evolution of the Ikaros Gene Family: Implications for the Origins of Adaptive Immunity

“…1 and 2). In sea squirt, two Ikaros-related (IFL) 4 The online version of this article contains supplemental material. proteins, designated IFL1 and IFL2, with homology with IFL sequences identified in other urochordates and other early vertebrates were discovered ( Fig.…”

“…Zinc finger transcription factors exhibit broad evolutionary conservation, including hematopoiesis. For example, GATA-related proteins regulate blood cell development from Drosophila to man (4,5).…”