Abstract
Epididymal retinoic acid-binding protein (ERABP) is the major androgen-dependent protein present in the lumen of the epididymis and is thought to be involved in sperm maturation. It displays a high degree of three-dimensional structural similarity to serum retinol-binding protein (RBP). Although both proteins interact with retinoids, RBP exhibits a broad specificity, binding retinol, retinoic acid and retinaldehyde with roughly equal affinities, whereas ERABP is specific for all-trans- and 9-cis-retinoic acids. Consistent with this, the binding pockets of the two proteins are different: in RBP it is predominantly hydrophobic, whereas that for ERABP is amphipathic, with a network of charged residues at the open end of the binding pocket. In order to investigate the roles of these charged residues, Arg-80 and Glu-63 have been mutated to isoleucine. The resultant double mutant, Glu-63-->Ile/Arg-80-->Ile, as well as the wild-type protein, were subsequently expressed in Escherichia coli as fusion proteins, with the streptavidin recognition sequence (Strep) tagged to their C-termini. The expressed proteins were purified in a single step by streptavidin-affinity chromatography and their ligand-binding properties were examined using fluorimetric titrations. Whereas the wild-type ERABP binds only retinoic acid, the double mutant is capable of binding retinol, retinoic acid and retinaldehyde with similar affinities. These observations provide experimental support for the proposition that the charged residues near the open end of the binding pocket are responsible for restricting the specificity of ERABP for retinoic acid. These studies demonstrate that changes in specificity can be engineered into lipocalins.
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Selected References
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