CELLULAR RETINOID-BINDING PROTEINS - LIMITED PROTEOLYSIS REVEALS A CONFORMATIONAL CHANGE UPON LIGAND-BINDING

Intracellular retinoid-binding proteins are small, tightly folded, com pact proteins, which appear to be involved in the delivery of retinoid s to microsomal metabolic enzymes, among other potential roles. Recent ly, it has been demonstrated that two of these binding proteins, cellu lar retinol-binding protein (CRBP) and cellular retinol-binding protei n type II [CRBP(II)], interact with the same microsomal enzyme but in different manners, depending on the absence or presence of ligand [Her r, F.M., & Ong, D.E. (1992) Biochemistry 31, 6748-6755]. The structura l components of the binding proteins responsible for these differentia l interactions are presently unknown. In addition, it is not clear how the ligand is able to gain entry into the solvent-inaccessible interi or binding cavity. Limited proteolysis of the apo and holo forms of CR BP and CRBP(II) was used to probe the conformational differences betwe en the different states of these two proteins in solution. It was foun d that the apo forms of both proteins were significantly more suscepti ble to proteolysis, and probably adopted a more open conformation, tha n the holo forms. The initial cleavage site of endoproteinase Arg-C in the apo forms occurred at a conserved arginine residue near a possibl e site of ligand entry. Similar results were obtained by limited prote olysis of cellular retinoic acid-binding protein and heart fatty acid- binding protein, indicating that a common ligand-induced conformationa l change may occur for other members of this family of intracellular b inding proteins. Additionally, it was found that the relative suscepti bility of holo-CRBP and holo-CRBP(II) to proteolysis was related to th eir affinities for ligand, with holo-CRBP, which has a significantly l ower Kd than that for holo-CRBP(II), being the more resistant to prote olysis. The results of the limited proteolysis experiments, and the im plications for protein-protein recognition and a ligand entry mechanis ms, can be related to the known X-ray structures of CRBP and CRBP(II).