Mapping the ligand binding pocket in the cellular retinaldehyde binding protein

Zhiping Wu, Yanwu Yang, Natacha Shaw, Sanjoy K Bhattacharya, Lin Yan, Karen West, Karen Roth, Noa Noy, Jun Qin, John W. Crabb

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)12390-12396
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number14
DOIs
StatePublished - Apr 4 2003
Externally publishedYes

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Retinaldehyde
Retinoids
Carrier Proteins
Ligands
Vitamin A
Substrates
Nuclear magnetic resonance
Phospholipid Transfer Proteins
Photoisomerization
Mutant Proteins
Isomerization
Kinetic parameters
Protein Binding
Amino Acid Sequence
Oxidation
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mapping the ligand binding pocket in the cellular retinaldehyde binding protein. / Wu, Zhiping; Yang, Yanwu; Shaw, Natacha; Bhattacharya, Sanjoy K; Yan, Lin; West, Karen; Roth, Karen; Noy, Noa; Qin, Jun; Crabb, John W.

In: Journal of Biological Chemistry, Vol. 278, No. 14, 04.04.2003, p. 12390-12396.

Research output: Contribution to journalArticle

Wu, Z, Yang, Y, Shaw, N, Bhattacharya, SK, Yan, L, West, K, Roth, K, Noy, N, Qin, J & Crabb, JW 2003, 'Mapping the ligand binding pocket in the cellular retinaldehyde binding protein', Journal of Biological Chemistry, vol. 278, no. 14, pp. 12390-12396. https://doi.org/10.1074/jbc.M212775200
Wu, Zhiping ; Yang, Yanwu ; Shaw, Natacha ; Bhattacharya, Sanjoy K ; Yan, Lin ; West, Karen ; Roth, Karen ; Noy, Noa ; Qin, Jun ; Crabb, John W. / Mapping the ligand binding pocket in the cellular retinaldehyde binding protein. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 14. pp. 12390-12396.
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