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Originally published In Press as doi:10.1074/jbc.M207300200 on January 20, 2003

J. Biol. Chem., Vol. 278, Issue 14, 12397-12402, April 4, 2003
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Disease-causing Mutations in the Cellular Retinaldehyde Binding Protein Tighten and Abolish Ligand Interactions*,

Irina Golovlevaab, Sanjoy Bhattacharyac, Zhiping Wucd, Natacha Shawe, Yanwu Yangf, Khurshid Andrabic, Karen A. Westc, Marie S. I. Burstedtg, Kristina Forsmanah, Gösta Holmgrena, Ola Sandgreng, Noa Noye, Jun Qinf, and John W. Crabbcdfi

From the Departments of a Medical Biosciences and g Ophthalmology, Umeå University, S-901 85 Umeå, Sweden, the c Cole Eye Institute, and f Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the e Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, and the d Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115

Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.

* This study was supported in part by National Institutes of Health Grants EY6603, EY14239, HL58758, and CA68150, by a Research Center grant from The Foundation Fighting Blindness, by funds from the Cleveland Clinic Foundation, and by grants from the Swedish Medical Research Council (Projects 10866 and 9745). A preliminary report of this work was presented at The Annual Meeting of the Association for Research in Vision and Ophthalmology, April 29 through May 4, 2001, Ft. Lauderdale, FL (35).The costs of publication of this article were defrayed in part by the payment of page charges. The 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 Fig. S1.

b To whom correspondence may be addressed: Clinical Genetics, Norrlands University Hospital, Umeå University, S 901 85 Umeå, Sweden. Tel.: 46-90-785-1781; Fax: 46-90-128-163; E-mail: irina.golovleva.us@vll.se.

h Present address: Dept. of Molecular Biology, Astra Zeneca, S-431 83 Mölndahl, Sweden.

i To whom correspondence may be addressed: Cole Eye Institute (i31), Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-0425; Fax: 216-445-3670; E-mail: crabbj@ccf.org.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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