Metal and RNA binding properties of the hdm2 RING finger domain

The hdm2 oncoprotein contains a C-terminal domain that binds RNA and has be en suggested to bind zinc(II) in an unusual RING finger domain in which Thr 455 was postulated as a ligand. We have reported experiments to test wheth er this C-terminal cysteine-rich motif is indeed a RING finger domain. We a lso tested the affinity of the hdm2 C-terminal peptide for metal binding, m etal linkage to the folding of the C-terminal peptide, and the peptide's af finity for RNA. Truncation mutants demonstrate that amino acids 425-491 are necessary and sufficient for RNA binding. However, divalent metal ions do not seem to affect the specific RNA recognition. Metal binding studies sugg est that hdm2 indeed binds to two molecules of zinc in an intertwined motif similar to the BRCA1 RING finger peptide. However, there is no similarity in overall tertiary structure, nor is there direct sequence homology with o ther RING fingers. Fluorescence energy transfer studies give a dissociation constant of (0.22 +/- 0.03) mu M for cobalt(II) binding to site 1, while K -2 for cobalt(II) binding was estimated to be 15 +/- 5 mu M from ultraviole t absorbance. Studies of two mutant peptides confirm the assignment of bind ing residues in hdm2 and suggest that the coordination of Thr 455 previousl y proposed by sequence alignments is incorrect. Structural studies of hdm2 in the presence and absence of metal indicate only a small amount of second ary structure by circular dichroic spectroscopy. Metal binding did not seem to nucleate folding as in the case of two other RING finger proteins. Howe ver, distance measurement from fluorescence energy transfer indicated that the Tyr 489 residue was only similar to 14 Angstrom away from the first met al center, suggesting that the hdm2 protein exists in a compact form, at le ast in the presence of metal ion. In summary, hdm2 binds metal and RNA, but the RNA binding does not seem to occur in a zinc-dependent manner.