Identification of CRALBP ligand interactions by photoaffinity labeling, hydrogen/deuterium exchange, and structural modeling.
Cellular retinaldehyde-binding protein (CRALBP) functions in the retinal pigment epithelium (RPE) as an acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a substrate carrier for 11-cis-retinol dehydrogenase. Toward a better understanding of CRALBP function, the ligand binding cavity in human recombinant CRALBP (rCRALBP) was characterized by photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal and by high resolution mass spectrometric topological analyses. Eight photoaffinity-modified residues were identified in rCRALBP by liquid chromatography tandem mass spectrometry, including Tyr(179), Phe(197), Cys(198), Met(208), Lys(221), Met(222), Val(223), and Met(225). Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown. Supporting the specificity of photo-labeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses. In addition, topological analysis of apo- and holo-rCRALBP by hydrogen/deuterium exchange and mass spectrometry demonstrated residues 198-255 incorporate significantly less deuterium when the retinoid binding pocket is occupied with 11-cis-retinal. This hydrophobic region encompasses all but one of the photo-labeled residues. A structural model of CRALBP ligand binding domain was constructed based on the crystal structures of three homologues in the CRAL-TRIO family of lipid-binding proteins. In the model, all of the photolabeled residues line the ligand binding cavity except Met(208), which appears to reside in a flexible loop at the entrance/exit of the ligand cavity. Overall, the results expand to 12 the number of residues proposed to interact with ligand and provide further insight into CRALBP ligand and protein interactions.
Wu
Zhiping
Z
Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Hasan
Azeem
A
Liu
Tianyun
T
Teller
David C
DC
Crabb
John W
JW
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006859:Hydrogen; D007536:Isomerism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012172:Retinaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D012176:Retinoids / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D014316:Tritium
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D008024:Ligands; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D012176:Retinoids; D014316:Tritium; D006859:Hydrogen; D012172:Retinaldehyde