title: Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions.
abstract: 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.
authors.affiliation: Department of Medical Biosciences, Umeå University, S-901 85 Umeå, Sweden. irena.golovleva.us@vll.se
authors.forename: Irina
authors.initials: I
authors.lastname: Golovleva
authors.forename: Sanjoy
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Wu
authors.forename: Natacha
authors.initials: N
authors.lastname: Shaw
authors.forename: Yanwu
authors.initials: Y
authors.lastname: Yang
authors.forename: Khurshid
authors.initials: K
authors.lastname: Andrabi
authors.forename: Karen A
authors.initials: KA
authors.lastname: West
authors.forename: Marie S I
authors.initials: MS
authors.lastname: Burstedt
authors.forename: Kristina
authors.initials: K
authors.lastname: Forsman
authors.forename: Gösta
authors.initials: G
authors.lastname: Holmgren
authors.forename: Ola
authors.initials: O
authors.lastname: Sandgren
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: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012176:Retinoids / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D008024:Ligands; D012176:Retinoids; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase