THE LIGAND-BINDING DOMAIN OF THE HUMAN RETINOIC ACID RECEPTOR-GAMMA IS PREDOMINANTLY ALPHA-HELICAL WITH A TRP RESIDUE IN THE LIGAND-BINDINGSITE

Retinoic acid exerts its many biological effects by interaction with a nuclear protein, the retinoic acid receptor (RAR). The details of thi s interaction are unknown due mainly to the lack of sufficient quantit ies of pure functional receptor protein for biochemical and structural studies. We have recently subcloned the D and E domains of human RAR gamma for expression in Escherichia coli. Using nickel-chelation affin ity chromatography with a polyhistidine amino terminal tail, purificat ion of the DE peptide with a pI of 5.18 was accomplished to greater th an 98% purity. Scatchard analysis and fluorescence quenching technique s using the purified protein indicate a very high percentage of functi onal molecules (>95%) with a K-d for retinoic acid (t-RA) of 0.6 +/- 0 .1 nM. Circular dichroism spectra of the purified domains predict a pr edominantly alpha-helical structure (similar to 56%) with little beta sheet present. No significant changes in these structural characterist ics were observed upon binding of t-RA. Inspection of the amino acid s equence within these domains identified a single tryptophan residue at position 227. Modeling the amino acid sequence in this region as an a lpha-helical structure indicates that this tryptophan is adjacent to a lanine 234, which corresponds to alanine 225 in RAR beta that has prev iously been linked to the ligand binding site. Fluorescence of this tr yptophan was quenched in a dose-dependent manner on the addition of t- RA, confirming that Trp-227 is within the ligand binding site. Tryptop han flourescence quenching analysis also demonstrates that a single re tinoic acid molecule is bound per receptor and suggests that receptor- ligand interactions occur within the amino-terminal portion of the pre dominantly alpha-helical ligand binding domain.