Metal binding properties and secondary structure of the zinc-binding domain of Nup475

Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens. Nup475 contains two tandemly repeated sequences YKTELCX₈CX₅CX₃H (Cys₃His repeats) that are thought to be zinc-binding domains. Similar sequences have been found in a number of proteins from various species of eukaryotes. To determine the metal binding properties and secondary structure of the putative zinc- binding domains of Nup475, we have used synthetic or recombinant peptides that contain one or two domain sequences. The peptide with a single domain bound 1.0 ± 0.1 equivalents of Co²⁺, and the peptide with two domains bound 1.7 ± 0.4 equivalents of Co²⁺. Both peptides bound Co²⁺ and Zn²⁺ with affinities similar to those of classical zinc finger peptides. In each case, the Co²⁺ complex exhibited strong d-d transitions characteristic of tetrahedral coordination. For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second Cys₃His repeat. The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first Cys₃His sequence. On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal- binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain. This structure is unlike that of any previously described class of metal binding domain.