ARGININE-132 OF CELLULAR RETINOIC ACID-BINDING PROTEIN (TYPE-II) IS IMPORTANT FOR BINDING OF RETINOIC ACID

Cellular retinoic acid binding protein type II (CRABP-II) is one of two small molecular weight, cytosolic proteins which specifically bind retinoic acid (RA). Crystallographic and site-directed mutagenesis studies of several related proteins have indicated that either one or two conserved amino acid residues, homologous to positions Arg(111) and Arg(132) of CRABP-II are important for the binding of the hydrophobic ligand. In this report we have prepared site-directed mutations of these two positions of CRABP-II, Arg(111) and Arg(132), as well as Lys(82) to determine the role of these residues in the binding of Rk Recombinant wild type and mutant CRABP-II proteins were expressed and purified, and the affinity for retinoids was determined by fluorometric titration and binding of H-3-labeled compounds. K82A displayed an identical K-d for all-trans-RA as wild type CRABP-II and the K-d for all-trans-RA of R111A was only slightly higher, On the other hand, the two Arg(132) mutants, R132A and R132Q, of CRABP-II demonstrated undetectable binding of all-trans-RA. Taken together these data demonstrate that Arg(132) is a critical amino acid residue for the binding of RA by CRABP-II.