Retinol binding sites

Occupancy of retinol binding site of transthyretin in vertebrate blood... 

RBP consists of a single globular domain composed of eight antiparallel )8 strands. Near the center of the )8 barrel, these strands enclose a cavity that is the retinol-binding site, as was shown in Fig. 2a. The rest of the secondary structure includes a short o helix and an additional /3 strand at the C terminus. The four-turn a helix packs near the /3 strands at an angle of about 15°. The side-chain interactions between the helix and /3 sheet are very extensive and involve mostly hydrophobic residues. 

Data analyses yield the number of retinol-binding sites per mole of protein (in our hands, 0 55-0 95 in different protein preparations), and the equilibrium dissociation constant (K ) of the retinol-IRBP complex (typically, 40-100 nM). 

These studies with retinoids have provided some information about the structural requirements of the retinol binding site on RBP. No information is, however, available about the amino acid residues in RBP that are involved in the binding site. Acetylation of lysine residues of RBP did not affect its binding of retinol (Heller and Horwitz, 1975). Modification of one of eight tyrosine residues and two of four tryptophan residues of RBP also had no effect on the retinol-RBP interaction (Heller and Horwitz, 1975 Horwitz and Heller, 1974b). The binding site was, however, disrupted by reduction and alkylation of disulfide bonds (Raz et al., 1970). 

The synthesis of a retinoid affinity label for the retinol binding site of RBP has been reported (Gawinowicz and Goodman, 1982). Three radioactive retinoid bromoacetates were synthesized as potential retinoid affinity labels. The H-labeled compounds synthesized were the bromoacetate derivatives of retinol... 

The interaction of retinol-CRBP with die nucleus has been further studied (Liau etal., 1981). Using CRBP labeled with tritium by reductive methylation, it was found that CRBP delivers retinol to the specific binding sites within the nucleus without itself remaining bound. The outer nuclear envelope is apparendy not involved because Triton X-lOO-treated nuclei retained 70% of the retinol binding sites found in intact nuclei. Isolated liver chromatin also had the same... 

The first example is the plasma-borne retinol-binding protein, RBP, which is a single polypeptide chain of 182 amino acid residues. This protein is responsible for transporting the lipid alcohol vitamin A (retinol) from its storage site in the liver to the various vitamin-A-dependent tissues. It is a disposable package in the sense that each RBP molecule transports only a single retinol molecule and is then degraded. 

Figure S.4 The binding site for retinol inside the RBP barrel is lined with hydrophobic residues. They provide a hydrophobic surrounding for the hydrophobic part of the retinol molecule.

Sequence-divergent units of the ABA-1 polyprotein array of the nematode Ascaris have similar fatty acid and retinol binding properties but different binding site environments. BiochemicalJournal340, 337-343. 

Altered vitamin A homeostasis, primarily manifested as decreased hepatic storage of vitamin A, is another established effect of PBBs in animals. Vitamin A is essential for normal growth and cell differentiation, particularly differentiation of epithelial cells, and some PBB-induced epithelial lesions resemble those produced by vitamin A deficiency. Because it is the primary storage site for vitamin A, the liver has a major role in retinol metabolism. Esterification of dietary vitamin A, hydrolysis of stored vitamin A, mobilization and release into the blood of vitamin A bound to retinol-binding protein, and much of the synthesis of retinol-binding protein occurs in the liver. 

Release from the liver When needed, retinol is released from fie liver and transported to extrahepatic tissues by the plasma retax -binding protein (RBP). The retinol-RBP complex attaches to spe cific receptors on the surface of the cells of peripheral tissues, permitting retinol to enter. Many tissues contain a cellular letaiol-binding protein that carries retinol to sites in the nucleus where the vitamin acts in a manner analogous to steroid hormones. 

Vitamin-E-binding sites have been demonstrated in a number of tissues. Catignani [240] has shown that the vitamin can bind to a cytoplasmic protein, with a molecular weight of about 31000, extracted from rat liver. Similar cytoplasmic proteins have been reported for 1,25-dihydroxycholecalciferol [241,242], retinol [243,244] and retinoic acid [245,246]. The vitamin-E-... 

Nakshatri, H., and Chambon, P. (1994). The Directly Repeated RG(G/T)TGA Motifs of the Rat and Mouse Cellular Retinol-Binding Protein II Genes Are Promiscuous Binding Sites for RAR, RXR, HNF-4, and ARP-1 Homo- and Heterodimers./. 

Metabolites of polychlorinated biphenyls bind to the thyroxine binding site of transthyretin and, in doing so, impair the binding of RBP. As a result of this, there is free RBP-bound retinol in plasma, which is filtered at the glomerulus andhence lost in the urine. This may account for the vitamin A depleting action of polychlorinated biphenyls (Brouwer and van den Berg, 1986). 

In the retinal pigment epithelium, palmitate is bound to the fatty acid binding site of the interphotoreceptor RBP, and the retinoid binding site has a high affinity for 11 -ds-retinaldehyde, which is to be transported to the photoreceptor cells. In the photoreceptor cells, the palmitate is displaced by docosahex-aenoic acid, which causes a conformational change in the protein, so that it no longer binds 11 -ds-retinaldehyde, which is delivered to the photoreceptor cells and binds all-fraws-retinol for transport back to the pigment epithelium. Here, the docosahexaenoic acid is displaced by palmitate, and the affinity of the protein for 11-ds-retinaldehyde is restored (Palczewski and Saari, 1997 Tschanz and Noy, 1997). 

Tschanz CL and Noy N (1997) Binding of retinol in both retinoid-binding sites of interphotoreceptor retinoid-binding protein (IRBP) is stabilized mainly by hydrophobic mtetActiom,. Journal of Biological Chemistry 272,30201-7. 

Mittag T, et al. Retinol modulates site-specific mobility of 118. apo-cellular retinol-binding protein to promote ligand binding. 

Stump, D. G., Lloyd, R. and Chytil, F. (1991). Site irected mutagenesis of rat cellular retinol-binding protein. /. Biol. Chem. 266,4622—4630. 

The accommodation of the hydrocarbon tail is the same in all but the insect form of the protein, MFB2, as shown in Fig. 13. If viewed in stereo, it should be apparent that all of the ligands are bent. Retinol has less curvature than the fatty acids. Although never analyzed in detail, the curvature occurs near the C-9 and C-10 positions of the fatty acid. The binding site therefore appears to be premade for unsaturated fatty acids. 

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