Biophysical characterization of zinc(II)-binding domains in two systems: Neural zinc finger factor-1 and T-cell co-receptors DC4 and DC8 alpha with T-cell-specific kinase Lck.

摘要

The first zinc(II)-binding domain described in transcription factors contains a CCHH motif. Transcription factors characterized by a CCHC zinc(II)-binding motif have also been identified, including Neural Zinc Finger Factor 1 (NZF-1). NZF-1 contains six zinc(II)-binding domains with a CCHHC motif. Domains two and three specifically bind the beta-retinoic acid response element DNA sequence. Previous NMR structural studies indicate the second conserved histidine of the motif is involved in stabilizing stacking interactions rather than metal binding. Substitution of phenylalanine for this histidine in a two-domain construct results in metal-binding affinity similar to wild type, but diminished DNA-binding affinity. In addition, comparison of the 1D 1H NMR spectra of the wild type and variant proteins reveals the protein does not fold properly without the second histidine. These results indicate the conserved non-metal-binding His must serve a structural role other than stacking. Lead(II) can bind to this metal site and disrupt DNA binding; however, the domain has at least two orders of magnitude less affinity for Pb(II) than Zn(II).; Recently, the "zinc clasp" was described (Kim, PW et al., Science , 301(5640), 19 Sep 2003, pp. 1725-1728). In this domain, two distinct proteins, each containing a dicysteine motif, coordinate Zn(II) in complex. The C-terminal cytosolic domains of T-cell co-receptors CD4 and CD8alpha have a CXC motif that interacts with the N-terminal CXXC motif of T-cell-specific Src-family kinase, Lck. While the heterodimer is involved in T-cell signaling, no evidence for homodimer formation has been reported. Size exclusion chromatography, 1D 1H and 19F NMR, and visible spectroscopy with Co(II) as a spectroscopic probe for Zn(II) show that each peptide does form a homodimer. Additional studies reveal heterodimers form preferentially to homodimers. The overall charge of the peptides and spacing between cysteines in the dicysteine motif were modified to study the effect on preferential heterodimer formation. Neither charge- nor cysteine spacing-modified peptides have preferential heterodimer formation relative to Lck homodimer formation. The Homo sapiens proteome was searched for similar motifs, but yielded no broad category of proteins utilizing this type of interaction.