Abstract
Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo- and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most relevant difference between the NMR structure ensembles of apo- and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligand-binding site. These comprise alpha-helix II, the subsequent linker to beta-strand B, the hairpin turn between beta-strands C and D, and the betaE-betaF turn. The internal backbone dynamics, obtained from 15N relaxation data (T1, T2, and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the betaC-betaD and betaE-betaF turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The 1H and 15N chemical shifts have been deposited in the BioMagResBank database under accession numbers 5048 and 5319 for apo- and holoCRBP, respectively, and the 15N relaxation data have been deposited in the BioMagResBank database under accession numbers 5330 and 5331 for apo- and holoCRBP, respectively
The poorly water-soluble retinol is stored within membranes as a retinyl-ester derivative of long-chain fatty acids, whose cellular retinolbinding protein type I (CRBP)-dependent synthesis is catalyzed by the microsomal enzyme lecithin:retinol acyltransferase [13]
Summary
Most stable complex with all-trans-retinol and a weaker one with retinaldehyde, whereas it does not bind retinoic acid [21]. A possible “portal” has been identified in the region located between ␣-helix II and the two turns C-D and E-F This hypothesis was initially based on the occurrence of a small opening on the surface of intestinal fatty acid-binding protein (I-FABP) [22]. The crystal structures of most i-LBPs reveal only minimal differences between the apo- and the holo-form This feature could in principle be attributed to lattice interactions that select the same protein conformation both in the presence and in the absence of the ligand, masking differences that possibly exist in solution. The solution structure of human apoCRABP-II shows that the binding cavity is readily accessible to retinoic acid, as a result of a concerted conformational change of ␣-helix II and the two turns
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