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

Irina Golovleva, Sanjoy K 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, John W. Crabb

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)12397-12402
Number of pages6
JournalJournal of Biological Chemistry
Volume278
Issue number14
DOIs
StatePublished - Apr 4 2003
Externally publishedYes

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Retinaldehyde
Retinoids
Carrier Proteins
Ligands
Mutation
Conformations
Substrates
Photoisomerization
Proteins
Fluorescence Spectrometry
Fluorescence spectroscopy
Pathology
Vitamin A
Solubility
Mass spectrometry
Mass Spectrometry
Genes
Nuclear magnetic resonance
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions. / Golovleva, Irina; Bhattacharya, Sanjoy K; Wu, Zhiping; Shaw, Natacha; Yang, Yanwu; Andrabi, Khurshid; West, Karen A.; Burstedt, Marie S I; Forsman, Kristina; Holmgren, Gösta; Sandgren, Ola; Noy, Noa; Qin, Jun; Crabb, John W.

In: Journal of Biological Chemistry, Vol. 278, No. 14, 04.04.2003, p. 12397-12402.

Research output: Contribution to journalArticle

Golovleva, I, Bhattacharya, SK, Wu, Z, Shaw, N, Yang, Y, Andrabi, K, West, KA, Burstedt, MSI, Forsman, K, Holmgren, G, Sandgren, O, Noy, N, Qin, J & Crabb, JW 2003, 'Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions', Journal of Biological Chemistry, vol. 278, no. 14, pp. 12397-12402. https://doi.org/10.1074/jbc.M207300200
Golovleva, Irina ; Bhattacharya, Sanjoy K ; Wu, Zhiping ; Shaw, Natacha ; Yang, Yanwu ; Andrabi, Khurshid ; West, Karen A. ; Burstedt, Marie S I ; Forsman, Kristina ; Holmgren, Gösta ; Sandgren, Ola ; Noy, Noa ; Qin, Jun ; Crabb, John W. / Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 14. pp. 12397-12402.
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AU - Golovleva, Irina

AU - Bhattacharya, Sanjoy K

AU - Wu, Zhiping

AU - Shaw, Natacha

AU - Yang, Yanwu

AU - Andrabi, Khurshid

AU - West, Karen A.

AU - Burstedt, Marie S I

AU - Forsman, Kristina

AU - Holmgren, Gösta

AU - Sandgren, Ola

AU - Noy, Noa

AU - Qin, Jun

AU - Crabb, John W.

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N2 - 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.

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