Plasma Retinol-Binding Protein: Structure and Interactions with Retinol, Retinoids, and Transthyretin
Introduction
Retinol (vitamin A alcohol) is an essential micronutrient, the active derivatives of which, such as 11-cis-retinal and all-trans- and 9-cis-retinoic acid, play key roles in a number of biological processes, including vision, cell growth and differentiation, embryonic development, and morphogenesis. Retinol is a chemically unstable and quite insoluble compound in the aqueous medium. Therefore, its interaction with binding proteins that protect and solubilize it appears to be crucial. In plasma and within the cell the retinol molecule is bound to distinct transport proteins, respectively, the plasma retinol-binding protein (RBP) and the cellular retinol-binding proteins (CRBPs). When complexed with its plasma and cytoplasmic carriers, retinol is bound inside a deep cavity, nearly completely shielded from the external environment, thereby acquiring a stability drastically higher than that of uncomplexed retinol.
RBP is a single polypeptide chain of 21 kDa, containing one binding site for all-trans-retinol. Goodman and colleagues (Kanai et al., 1968) first reported its isolation from human serum, and since then RBP has been isolated from the serum of a number of other vertebrates, including birds and fish. This binding protein, which is uniquely involved in the transport of retinol in the circulation (Goodman, 1984), is synthesized primarily in the liver, the major storage site of vitamin A, and is secreted as the retinol–RBP complex into the circulation, where it transports the vitamin to be delivered to peripheral target tissues. The availability of retinol plays a critical role in the secretion of RBP by hepatocytes. In fact, the secretion of RBP is specifically blocked by retinol deficiency and is rapidly restored by retinol repletion (Goodman 1984, Ronne 1983).
Holo-RBP is present in the blood of higher vertebrates almost entirely bound to another plasma protein, transthyretin (TTR). TTR is a homotetramer of 55 kDa involved, together with other plasma proteins, in the transport of thyroid hormones (L-thyroxine⧸triiodo-L-thyronine) (for a review on the thyroid hormone transport properties of TTR, see Palha, 2002). This protein forms in vivo a moderately tight 1:1 molar complex with RBP. The association of holo-RBP with TTR increases the stability of the retinol–RBP complex (Goodman, 1984). Various lines of evidence indicate that the association of holo-RBP with TTR also serves to prevent filtration of the relatively small RBP molecule through kidney glomeruli (Episkopou 1993, Goodman 1984, van Bennekum 2001). The stability of the RBP–TTR complex is strongly affected by the presence of retinol bound to RBP within the complex, a feature which is believed to be of physiological significance. In fact, the affinity of holo-RBP for TTR has been found to be significantly higher than that of apo-RBP by the use of different methodologies (Goodman 1984, Malpeli 1996, Zanotti 1993a). The different affinity is consistent with holo-RBP being retained in the circulation as the protein–protein complex (76 kDa) and with the uncomplexed and relatively small apo-RBP molecule (21 kDa), resulting from the delivery of retinol, being selectively cleared from the circulation by glomerular filtration.
RBP belongs to a superfamily of proteins, known as the lipocalin superfamily, the members of which possess diverse functions and are characterized by a highly conserved structural motif, despite the fact that they may share a low degree of sequence identity. They have been designated “lipocalins,” owing to the fact that their structure has the shape of a calyx and that their ligands are principally hydrophobic compounds. RBP can be considered to be the prototypic lipocalin, having been the first lipocalin for which the crystal structure was described (Newcomer et al., 1984). A considerable amount of information has been obtained by the analysis of a number of crystal structures of RBP: holo-RBP (Calderone 2003, Cowan 1990, Newcomer 1984, Zanotti 1993a, Zanotti 1993c); apo-RBP (Greene 2001, Zanotti 1993a, Zanotti 1993c); holo-RBP at different pH values (Calderone et al., 2003); RBP from different species (Cowan 1990, Zanotti 1993a, Zanotti 1993a, Zanotti 1998, Zanotti 2001); RBP in complex with retinoids (Zanotti 1993b, Zanotti 1994); RBP present in a macromolecular complex, associated with TTR (Monaco 1995, Naylor 1999); a mutant form of RBP, in which two conserved W residues were replaced (Greene et al., 2001). These structures, which are summarized in Table I, are all quite consistent, albeit obtained for RBPs from different vertebrate species and crystallized in different conditions and space groups. Overall, they can thus be considered to be well representative of the protein structure in solution. A representation of the RBP structure is shown in Fig. 1. This single-domain protein is made up of a N-terminal coil, eight antiparallel β-strands (A–H), and a short α-helix close to the C terminus. The core of the protein is an eight-stranded up-and-down β-barrel, which forms a cavity where the ligand binds. The retinol molecule is encapsulated inside this cavity, with the cyclohexene ring, which is innermost, and the polyene chain completely buried. The alcoholic group of the vitamin is nearly at the protein surface, in the region of the entrance loops, which surround the entrance of the β-barrel at the open end of the cavity. Multiple amino acid sequence alignments for RBPs and TTRs from phylogenetically distant vertebrate species, showing the position of secondary structure elements of the two proteins and of interacting amino acid residues at the interface between the contact areas of RBP and TTR, are presented in Fig. 2. Here, we describe structural properties of RBP in connection with its interactions with small-size ligands and TTR and biochemical implications of molecular interactions.
Section snippets
High-Resolution Structure of Retinol-Binding Protein in Complex with Retinol
The models of bovine holo-RBP determined recently at different pH values (Calderone et al., 2003) represent the highest resolution (1.1–1.5 Å, depending on the pH value of the crystal medium) crystal structures of RBP determined so far. We report here on some relevant features of these structures.
The electron density for all-trans-retinol within the β-barrel internal cavity is very well defined. The conformation of the cyclohexene ring of bound retinol is that of a half-chair, in agreement with
Structure of Apo Retinol-Binding Protein
Apo-RBP suitable for the preparation of single protein crystals has been obtained by depleting plasma holo-RBP of retinol with the use of a chromatographic procedure (Zanotti et al., 1993c) or of an organic solvent (Zanotti et al., 1993a) or by the heterologous expression in Escherichia coli of human apo-RBP (Greene et al., 2001). These procedures do not lead to the collapse of the β-barrel. In fact, the extraction of retinol from the cavity of both human and bovine holo-RBPs does not cause any
Structure of Retinol-Binding Protein in Complex with Retinoids
Retinoids constitute a large group of naturally occurring or synthetic compounds related structurally to retinol. The great interest for these compounds and for their biological activities raises relevant questions as to their possible interactions with retinoid-binding proteins, enzymes, and retinoid receptors. Synthetic retinoids may interact in vivo with retinoid-binding proteins and enzymes involved in the metabolism of vitamin A, thus interfering with the transport and metabolism of
Structure of the Retinol-Binding Protein–Transthyretin Complex
TTR physiologically binds thyroid hormones and forms a protein–protein complex with RBP in higher vertebrates. TTR is composed of four identical subunits of approximately 14 kDa, consisting mainly of two four-stranded β-sheets, which are assembled around a central channel containing two nearly identical thyroid hormone-binding sites (Blake 1978, Hornberg 2000). A large number of point mutations in human TTR, which promote the formation of amyloid fibrils containing mutated TTR as the major
Summary and Conclusions
RBP mediates the transport of vitamin A alcohol from its main storage site, the liver, to peripheral target tissues, solubilizing and protecting the vitamin, which is bound inside a deep cavity in the retinol–RBP complex. A high degree of conservation of the three-dimensional structures of phylogenetically distant RBPs from higher vertebrates, which is attributable to the presence in the RBP molecule of molecular constraints imposed by its multiple interactions with retinol, TTR, and possibly
Acknowledgements
Some of the data sets mentioned in this chapter were measured at the synchrotron facilities of Elettra (Trieste, Italy) and ESRF (Grenoble, France). We are grateful to Claudia Folli and Ileana Ramazzina for the help with the preparation of the manuscript. This study was supported by the National Research Council of Italy (to G. Z.) and MIUR Cofin Projects (to G. Z. and R. B.).
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2020, Protein Expression and PurificationCitation Excerpt :Similarly, the peak at 196 nm and the well at 208 nm match the spectra of serum RBP. The high β-sheet content CD spectra also agrees with the expected tertiary structure based on apo and holo RBP crystal structures [1], which indicate serum apo/holo RBP form a β-barrel structure motif. Next we evaluated the stability of rRBP against chemical denaturation, and the effect of ligands RA and ROH on stability.