title: Mapping the ligand binding pocket in the cellular retinaldehyde binding protein.
abstract: 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.
authors.affiliation: Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Wu
authors.forename: Yanwu
authors.initials: Y
authors.lastname: Yang
authors.forename: Natacha
authors.initials: N
authors.lastname: Shaw
authors.forename: Sanjoy
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Lin
authors.initials: L
authors.lastname: Yan
authors.forename: Karen
authors.initials: K
authors.lastname: West
authors.forename: Karen
authors.initials: K
authors.lastname: Roth
authors.forename: Noa
authors.initials: N
authors.lastname: Noy
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
authors.forename: John W
authors.initials: JW
authors.lastname: Crabb
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012176:Retinoids / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D008024:Ligands; D012176:Retinoids