Identification of CRALBP Ligand Interactions by Photoaffinity Labeling, Hydrogen/Deuterium Exchange, and Structural Modeling*□S

Cellular retinaldehyde-binding protein (CRALBP) functions in the retinal pigment epithelium (RPE) as an acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a substrate carrier for 11-cisretinol dehydrogenase. Toward a better understanding of CRALBP function, the ligand binding cavity in human recombinant CRALBP (rCRALBP) was characterized by photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal and by high resolution mass spectrometric topological analyses. Eight photoaffinity-modified residues were identified in rCRALBP by liquid chromatography tandem mass spectrometry, including Tyr, Phe, Cys, Met, Lys, Met, Val, and Met. Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown. Supporting the specificity of photolabeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses. In addition, topological analysis of apoand holorCRALBP by hydrogen/deuterium exchange and mass spectrometry demonstrated residues 198–255 incorporate significantly less deuterium when the retinoid binding pocket is occupied with 11-cis-retinal. This hydrophobic region encompasses all but one of the photolabeled residues. A structural model of CRALBP ligand binding domain was constructed based on the crystal structures of three homologues in the CRAL-TRIO family of lipid-binding proteins. In the model, all of the photolabeled residues line the ligand binding cavity except Met, which appears to reside in a flexible loop at the entrance/exit of the ligand cavity. Overall, the results expand to 12 the number of residues proposed to interact with ligand and provide further insight into CRALBP ligand and protein interactions. Cellular retinaldehyde-binding protein (CRALBP) is a 36kDa water soluble protein with a high affinity binding pocket for retinoids uniquely associated with vision, namely 11-cisretinal and 11-cis-retinol (1). Human CRALBP gene defects can either tighten or abolish retinoid interactions, which in turn can compromise substrate carrier interactions with 11-cis-retinol dehydrogenase and lead to several retinal pathologies (2). Targeted disruption of the CRALBP gene in mice impairs regeneration of both rod and cone visual pigments (3). These and other studies establish multiple functions for CRALBP in the retinal pigment epithelium (RPE), including roles as a major acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a facilitator of oxidation of 11-cis-retinol to 11-cis-retinal by 11-cis-retinol dehydrogenase (2–6). Ongoing proteomic studies support the existence of a RPE retinoid processing protein complex containing CRALBP (7). Direct CRALBP interactions have been demonstrated in vitro with 11-cis-retinol dehydrogenase (2) and with ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50), also known as sodium hydrogen exchanger regulator factor type 1 (NHERF-1) (8). Interactions with EBP50 have been suggested as a mechanism for localizing CRALBP to the apical RPE plasma membrane for export of 11-cis-retinal to the adjacent rod photoreceptor cells for visual pigment regeneration (8). The functions of CRALBP in tissues other than the RPE remain to be determined (i.e. in retinal Müller cells, ciliary epithelium, iris, cornea, pineal gland, and a subset of oligodendrocytes of the optic nerve and brain). To better understand CRALBP visual cycle functions, which require rapid association and dissociation of retinoid, we are characterizing the structure of the ligand binding pocket. Ligand interactions in CRALBP are non-covalent and previous structure-function studies (2, 9, 10) have implicated eight residues as possibly interacting with retinoid (Fig. 1). In this report, the interaction between human recombinant CRALBP and retinoid were characterized by mass spectrometry using hydrogen/deuterium exchange and photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal (DK-11-cis-retinal). This photoaffinity analogue of 11-cis-retinal has previously been used to map the movement of ligand within rhodopsin following sensi* This study was supported in part by National Institutes of Health Grants EY6603, EY14239, GM63020, a Research Center Grant from The Foundation Fighting Blindness, and funds from the Cleveland Clinic Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1–4 and Supplemental Tables I and II. ¶ This work was submitted in partial fulfillment of the requirements for a doctoral degree in chemistry from Cleveland State University. Present address: University of Florida, General Clinical Research Center, Gainesville, FL 32610-0322. §§ To whom correspondence should be addressed: Cole Eye Institute (i31), Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, Ohio 44195. Tel.: 216-445-0425; Fax: 216-445-3670; E-mail: [email protected]. 1 The abbreviations used are: CRALBP, cellular retinaldehyde-binding protein; DK-11-cis-retinal, 3-diazo-4-keto-11-cis-retinal; DKrCRALBP, rCRALBP photolabeled with DK-11-cis-retinal; H/D exchange, hydrogen/deuterium exchange; LC ESMS, liquid chromatography electrospray mass spectrometry; LC MS/MS, liquid chromatography tandem mass spectrometry; QTOF, quadrupole time of flight; RPE, retinal pigment epithelium; rCRALBP, recombinant CRALBP; RP-HPLC, reverse phase-high performance liquid chromatography. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 26, Issue of June 25, pp. 27357–27364, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.