STRUCTURE-FUNCTION-RELATIONSHIPS OF CELLULAR RETINOIC ACID-BINDING PROTEINS - QUANTITATIVE-ANALYSIS OF THE LIGAND-BINDING PROPERTIES OF THEWILD-TYPE PROTEINS AND SITE-DIRECTED MUTANTS

It has been suggested that electrostatic interactions are critical for binding of retinoic acid by cellular retinoic acid-binding proteins ( CRABP-I and CRABP-II). However, the roles of two conserved arginine re sidues (Arg-111 and Arg-131 in CRABP-I; Arg-111 and Arg-132 in CRABP-I I) that interact with the carboxyl group of retinoic acid have not bee n evaluated. A novel competitive binding assay has been developed for measuring the relative dissociation constants of the site-directed mut ants of CRABPs. Arg-111 and Arg-132 of CRABP-II were replaced with met hionine by site-directed mutagenesis. The relative dissociation consta nts of R111M and R132M (K-d (R111M)/K-d (CRABP-II) and K-d (R132M)/K-d (CRABP-II)) were determined to be 40-45 and 6-8, respectively. The ri ng protons of the aromatic residues of the wild-type CRABP-II and the two mutants were sequentially assigned by two-dimensional homonuclear NMR in conjunction with three-dimensional heteronuclear NMR. Detailed analysis of the nuclear Overhauser effect spectroscopy spectra of the proteins indicated that the conformations of the two mutants are highl y similar to that of the wild-type CRABP-II. These results taken toget her showed that Arg-111 and Arg-132 are important for binding retinoic acid but contribute to the binding energy only by similar to 2.2 and 1.2 kcal/mol, respectively. In addition, the relative dissociation con stant of CRABP-II and CRABP-I (K-d (CRABP-II)/K-d (CRABP-I)) was deter mined to be 2-3, in close agreement with that calculated using the app arent K-d values determined under the same conditions by fluorometric titrations.