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A more recent version of this article appeared on March 28, 2003
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278/14/12397    most recent
M207300200v1
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Papers In Press, published online ahead of print January 20, 2003
J. Biol. Chem, 10.1074/jbc.M207300200
Submitted on July 19, 2002
Revised on January 16, 2003
Accepted on January 20, 2003

Disease causing mutations in the cellular retinaldehyde-binding protein tighten as well as abolish ligand interactions

Irina Golovleva, Sanjoy Bhattacharya, Zhiping Wu, Natacha Shaw, Yanwu Yang, Khurshid Andrabi, Karen A. West, Marie S. I. Burstedt, Kristina Forsman, Gösta Holmgren, Ola Sandgren, Noa Noy, Jun Qin, and John W. Crabb

Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH 44195

Corresponding Author: crabbj{at}ccf.org

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 wildtype rCRALBP and bound stoichiometric amounts of 11-cis and 9-cis-retinal with at least two-fold higher affinity than wildtype rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wildtype rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wildtype 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 wildtype 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.


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