CELLULAR RETINALDEHYDE-BINDING PROTEIN LIGAND INTERACTIONS - GLN-210 AND LYS-221 ARE IN THE RETINOID-BINDING POCKET

Cellular retinaldehyde-binding protein (CRALBP) carries Il-cis-retinal and/or Il-cis-retinol as endogenous ligands in the retinal pigment ep ithelium (RPE) and Muller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa, Ligand interactions determi ne the physiological role of CRALBP in the RPE where the protein is th ought to function as a substrate carrier for Il-cis-retinol dehydrogen ase in the synthesis of Il-cis-retinal for visual pigment regeneration . However, CRALBP is also present in optic nerve and brain where its n atural ligand and function are not yet known. We have characterized th e interactions of retinoids with native bovine CRALBP, human recombina nt CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or i dentify a Schiff base in the dark, under a variety of reducing, denatu ring, and pH conditions were unsuccessful, suggesting the lack of cova lent interactions between CRALBP and retinoid. Buried and solvent-expo sed lysine residues were identified in bovine CRALBP by reductive meth ylation of the holoprotein followed by denaturation and reaction with [H-3]acetic anhydride, Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteol ysis and purification of modified peptides, Human rCRALBP mutants K152 A, K221A, and K294A were prepared to investigate possible retinoid int eractions with buried or partially buried lysines. Two other rCRALBP m utants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The struc tures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by W-visible and f luorescence spectroscopy. AU of the mutants bound Il-cis-retinal essen tially like the wild type protein, indicating that the proteins were n ot grossly misfolded, Three of the mutants bound 9-cis-retinal like th e wild type protein; however, Q210R and K221A bound less than stoichio metric amounts of the g-cis-isomer and exhibited lower affinity for th is retinoid relative to wild type rCRALBP. Residues Gin-210 and Lys-22 1 are located within a region of CRALBP exhibiting sequence homology w ith the ligand binding cavity of yeast phosphatidylinositol-transfer p rotein, The data implicate Gin-210 and Lys-221 as components of the CR ALBP retinoid binding cavity and are discussed in the context of ligan d interactions in structurally or functionally related proteins with k nown crystallographic structures.