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J. Biol. Chem., Vol. 279, Issue 26, 27357-27364, June 25, 2004

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

Zhiping Wu¶, Azeem Hasan||, Tianyun Liu**, David C. Teller**, and John W. Crabb

From the Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, and the ^**Department of Biochemistry, University of Washington, Seattle, Washington 98195

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-cis-retinol 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 Tyr¹⁷⁹, Phe¹⁹⁷, Cys¹⁹⁸, Met²⁰⁸, Lys²²¹, Met²²², Val²²³, and Met²²⁵. Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown. Supporting the specificity of photo-labeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses. In addition, topological analysis of apo- and holo-rCRALBP 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 photo-labeled 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 Met²⁰⁸, 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.

Received for publication, February 23, 2004 , and in revised form, April 5, 2004.

^* 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.

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: crabbj{at}ccf.org.

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