title: Retinol-binding site in interphotoreceptor retinoid-binding protein (IRBP): a novel hydrophobic cavity.
abstract: OBJECTIVE
  Interphotoreceptor retinoid-binding protein (IRBP) appears to target and protect retinoids during the visual cycle. X-ray crystallographic studies had noted a betabetaalpha-spiral fold shared with crotonases and C-terminal protein transferases. The shallow cleft formed by the fold was assumed to represent the retinol-binding site. However, a second hydrophobic site consisting of a highly restricted cavity was more recently appreciated during in silico ligand-docking studies. In this study, the ligand-binding environment within the second module of Xenopus IRBP (X2IRBP) is defined.
  
  
  METHODS
  Pristine recombinant polypeptide corresponding to X2IRBP was expressed in a soluble form and purified to homogeneity without its fusion tag. Phenylalanine was substituted for tryptophan at each of the putative retinol-binding domains (W450F, hydrophobic cavity; W587F, shallow cleft). Binding of 11-cis and all-trans retinol were observed in titrations monitoring retinol fluorescence enhancement, quenching of tryptophan fluorescence, and energy transfer. The effect of oleic acid on retinol binding was also examined.
  
  
  RESULTS
  A ligand-binding stoichiometry of approximately 1:1 was observed for 11-cis and all-trans with K(d) in the tens of nanomolar range. The substitution mutants showed little effect on retinol binding in titrations after fluorescence enhancement. However, the W450F and not the W587F mutant showed a markedly reduced capacity for fluorescence quenching for both 11-cis and all-trans retinol. Oleic acid inhibited the binding of 11-cis and all-trans retinol in an apparent noncompetitive manner.
  
  
  CONCLUSIONS
  The binding site for 11-cis and all-trans retinol is a novel hydrophobic cavity that is highly restrictive and probably distinct from the long chain fatty acid-binding site.
authors.affiliation: Neuroscience Graduate Program, State University of New York at Buffalo, Buffalo, New York, USA. fg23@buffalo.edu
authors.forename: Federico
authors.initials: F
authors.lastname: Gonzalez-Fernandez
authors.forename: Thomas
authors.initials: T
authors.lastname: Bevilacqua
authors.forename: Kee-Il
authors.initials: KI
authors.lastname: Lee
authors.forename: Reena
authors.initials: R
authors.lastname: Chandrashekar
authors.forename: Larson
authors.initials: L
authors.lastname: Hsu
authors.forename: Mary Alice
authors.initials: MA
authors.lastname: Garlipp
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Griswold
authors.forename: Rosalie K
authors.initials: RK
authors.lastname: Crouch
authors.forename: Debashis
authors.initials: D
authors.lastname: Ghosh
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D019301:Oleic Acid / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014801:Vitamin A / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis; D000099214:Retinol-Binding Proteins, Interstitial
chemical_list: D005136:Eye Proteins; D008024:Ligands; D019301:Oleic Acid; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D014801:Vitamin A; D000099214:Retinol-Binding Proteins, Interstitial