A class of zinc fingers involved in protein-protein interactions - Biophysical characterization of CCHC fingers from Fog and U-shaped

Zinc fingers (ZnFs) are extremely common protein domains. Several classes o f ZnFs are distinguished by the nature and spacing of their zinc-coordinati ng residues. While the structure and function of some ZnFs are well charact erized, many others have been identified only through their amino acid sequ ence. A number of proteins contain a conserved C-X-2-C-X-12-H-X1-5-C sequen ce, which is similar to the spacing observed for the 'classic' CCHH ZnFs. A lthough these domains have been implicated in protein-protein (and not prot ein-nucleic acid) interactions, nothing is known about their structure or f unction at a molecular level. Here, we address this problem through the exp ression and biophysical characterization of several CCHC-type zinc fingers from the erythroid transcription factor FOG and the related Drosophila prot ein U-shaped. Each of these domains does indeed fold in a zinc-dependent fa shion, coordinating the metal in a tetrahedral manner through the sidechain s of one histidine and three cysteine residues, and forming extremely therm ostable structures. Analysis of CD spectra suggests an overall fold similar to that of the CCHH fingers, and indeed a point mutant of FOG-F1 in which the final cysteine residue is replaced by histidine remains capable of fold ing. However, the CCHC (as opposed to CCHH) motif is a prerequisite for GAT A-1 binding activity, demonstrating that CCHC and CCHH topologies are not i nterchangeable. This demonstration that members of a structurally distinct subclass of genuine zinc finger domains are involved in the mediation of pr otein-protein interactions has implications for the prediction of protein f unction from nucleotide sequences.