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J. Biol. Chem., Vol. 278, Issue 14, 12390-12396, April 4, 2003
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From the Retinoid interactions determine the
function of the cellular retinaldehyde binding protein (CRALBP) in the
rod visual cycle where it serves as an 11-cis-retinol
acceptor for the enzymatic isomerization of all-trans- to
11-cis-retinol and as a substrate carrier for
11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues,
human recombinant CRALBP (rCRALBP) with altered Met or Trp were
produced and analyzed for ligand interactions. The primary structures
of the purified proteins were verified for mutants M208A, M222A, M225A,
W165F, and W244F, then retinoid binding properties and substrate
carrier functions were evaluated. All the mutant proteins bound
11-cis- and 9-cis-retinal and therefore were
not grossly misfolded. Altered UV-visible spectra and lower retinoid
binding affinities were observed for the mutants, supporting modified
ligand interactions. Altered kinetic parameters were observed for RDH5
oxidation of 11-cis-retinol bound to rCRALBP mutants M222A,
M225A, and W244F, supporting impaired substrate carrier function.
Heteronuclear single quantum correlation NMR analyses confirmed
localized structural changes upon photoisomerization of
rCRALBP-bound 11-cis-retinal and
demonstrated ligand-dependent conformational changes for
residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore,
residues Met-208, Met-222, Met-225, and Trp-244 are within a region
exhibiting high homology to the ligand binding cavity of
phosphatidylinositol transfer protein. Overall the data implicate
Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the
CRALBP ligand binding cavity.
Mapping the Ligand Binding Pocket in the Cellular
Retinaldehyde Binding Protein*,
§,
,,,,,,§¶§§
Cole Eye Institute and ¶ Lerner
Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the § Department of Chemistry, Cleveland State University,
Cleveland, Ohio 44115, the Division of Nutritional Sciences,
Cornell University, Ithaca, New York 14853, and the ** former Adirondack
Biomedical Research Institute, Lake Placid, New York
12946
*
This work 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, and by funds from
the Cleveland Clinic Foundation. 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 (28).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 Figs. S1-S3 and Supplemental
Table SI.
Present address: LaSalle School, 391 Western Ave. Albany, NY 12203.
§§
To whom correspondence should 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.
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