CRYSTAL-STRUCTURE OF APO-CELLULAR RETINOIC ACID-BINDING PROTEIN TYPE-II (R111M) SUGGESTS A MECHANISM OF LIGAND ENTRY

The crystal structure of unliganded mutant R111M of human cellular ret inoic acid-binding protein type II (apo-CRABPII (R111M)) has been dete rmined at 2.3 Angstrom and refined fro a crystallographic R-factor of 0.18. Although the mutant protein has lower affinity for all-trans-ret inoic acid (RA) than the wild-type, it is properly folded, and its con formation is very similar to the wild-type. apo-CRABPII (R111M) crysta llizes in space group P1 with two molecules in the unit cell. The two molecules have high structural similarity except that their alpha 2 he lices differ strikingly. Analyses of the molecular conformation and cr ystal packing environment suggest that one of the two molecules assume s a conformation compatible with RA entry. Three structural elements e ncompassing the opening of the binding pocket exhibit large conformati onal changes, when compared with holo-CRABPII, which include the alpha 2 helix and the beta C-beta D and beta E-beta F hairpin loops. The al pha 2 helix is unwound at its N terminus, which appears to be essentia l for the opening of the RA-binding pocket. Three arginine side-chains (29, 59, and 132) are found with their guanidino groups pointing into the RA-binding pocket. A three-step mechanism of TCA entry has been p roposed, addressing the opening of the RA entrance, the electrostatic potential that directs entry of RA into the binding pocket, and the in tramolecular interactions that stabilize the RA.CRABPII complex via lo cking the three flexible structural elements when RA is bound. (C) 199 8 Academic Press Limited.