Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions.
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.
Golovleva
Irina
I
Department of Medical Biosciences, Umeå University, S-901 85 Umeå, Sweden. irena.golovleva.us@vll.se
Bhattacharya
Sanjoy
S
Wu
Zhiping
Z
Shaw
Natacha
N
Yang
Yanwu
Y
Andrabi
Khurshid
K
West
Karen A
KA
Burstedt
Marie S I
MS
Forsman
Kristina
K
Holmgren
Gösta
G
Sandgren
Ola
O
Noy
Noa
N
Qin
Jun
J
Crabb
John W
JW
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
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D008024:Ligands; D012176:Retinoids; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase