The ligand binding domain of the human retinoic acid receptor γ is predominantly α-helical with a Trp residue in the ligand binding site

Abstract

Retinoic acid exerts its many biological effects by interaction with a nuclear protein, the retinoic acid receptor (RAR). The details of this interaction are unknown due mainly to the lack of sufficient quantities of pure functional receptor protein for biochemical and structural studies. We have recently subcloned the D and E domains of human RARγ for expression in Escherichia coli. Using nickel-chelation affinity chromatography with a polyhistidine amino-terminal tail, purification of the DE peptide with a pI of 5.18 was accomplished to greater than 98% purity. Scatchard analysis and fluorescence quenching techniques using the purified protein indicate a very high percentage of functional 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 predominantly α-helical structure (~56%) with little β sheet present. No significant changes in these structural characteristics were observed upon binding of t-RA. Inspection of the amino acid sequence within these domains identified a single tryptophan residue at position 227. Modeling the amino acid sequence in this region as an α-helical structure indicates that this tryptophan is adjacent to alanine 234, which corresponds to alanine 225 in RARβ that has previously been linked to the ligand binding site. Fluorescence of this tryptophan was quenched in a dose-dependent manner on the addition of t-RA, confirming that Trp-227 is within the ligand binding site. Tryptophan flourescence quenching analysis also demonstrates that a single retinoic acid molecule is bound per receptor and suggests that receptor-ligand interactions occur within the amino-terminal portion of the predominantly α-helical ligand binding domain.