Ja. Lupisella et al., THE LIGAND-BINDING DOMAIN OF THE HUMAN RETINOIC ACID RECEPTOR-GAMMA IS PREDOMINANTLY ALPHA-HELICAL WITH A TRP RESIDUE IN THE LIGAND-BINDINGSITE, The Journal of biological chemistry, 270(42), 1995, pp. 24884-24890
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.