Title

Cellular Retinoid-Binding Proteins: Limited Proteolysis Reveals a Conformational Change upon Ligand Binding

Document Type

Article

Publication Date

3-1-1994

Abstract

Intracellular retinoid-binding proteins are small, tightly folded, compact proteins, which appear to be involved in the delivery of retinoids to microsomal metabolic enzymes, among other potential roles. Recently, it has been demonstrated that two of these binding proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein type II [CRBP(II)], interact with the same microsomal enzyme but in different manners, depending on the absence or presence of ligand [Herr, F. M., & Ong, D. E. (1992) Biochemistry 31, 6748–6755]. The structural components of the binding proteins responsible for these differential interactions are presently unknown. In addition, it is not clear how the ligand is able to gain entry into the solvent-inaccessible interior binding cavity. Limited proteolysis of the apo and holo forms of CRBP and CRBP(II) was used to probe the conformational differences between the different states of these two proteins in solution. It was found that the apo forms of both proteins were significantly more susceptible to proteolysis, and probably adopted a more open conformation, than the holo forms. The initial cleavage site of endoproteinase Arg-C in the apo forms occurred at a conserved arginine residue near a possible site of ligand entry. Similar results were obtained by limited proteolysis of cellular retinoic acid-binding protein and heart fatty acid-binding protein, indicating that a common ligand-induced conformational change may occur for other members of this family of intracellular binding proteins. Additionally, it was found that the relative susceptibility of holo-CRBP and holo-CRBP(II) to proteolysis was related to their affinities for ligand, with holo-CRBP, which has a significantly lower Kd than that for holo-CRBP(II), being the more resistant to proteolysis. The results of the limited proteolysis experiments, and the implications for protein-protein recognition and ligand entry mechanisms, can be related to the known X-ray structures of CRBP and CRBP(II). © 1994, American Chemical Society. All rights reserved.

Publication Source (Journal or Book title)

Biochemistry

First Page

2873

Last Page

2879

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