Structure
Research ArticleCrystal structures of cellular retinoic acid binding proteins I and II in complex with all-trans-retinoic acid and a synthetic retinoid
Introduction
Vitamin A (retinol) and its active metabolite retinoic acid (RA) have profound effects on cell growth and differentiation. High doses of RA induce malformations in developing embryos, including limb abnormalities and facial defects. These retinoids have found use in a number of medical applications including the treatment of some cancers [1] and skin disorders [2].
The biological activities of RA appear to be mediated by nuclear receptors, retinoic-acid receptors (RAR) and retinoid X receptors (RXR), that modulatethe transcriptional activity of specific genes in a ligand-dependent fashion [3], [4], [5]. These receptors are members of a larger group, the steroid and thyroid hormone nuclear receptor superfamily, and are built up from protein modules that include DNA-binding and ligand-binding domains. The structures of RAR and RXR are unknown at present, with the exception of the DNA-binding domains of RAR-β [6] and RXR-α [7]. A second family of cellular retinoid-binding proteins has been identified that specifically bind either retinol (CRBPs) or RA (CRABPs). Two forms of CRABP have been identified, types I and II [8], [9], and these have different properties [10] and levels of expression, both in the adult and during development [11], [12], [13].
CRABP I shows somewhat higher binding affinity for RA than does CRABP II [10], [14]. The amino acid sequence of CRABP I is highly conserved among species, showing only one residue change upon alignment of human, bovine and murine sequences. CRABP II displays larger sequence variation but is still highly conserved; the full-length human and murine sequences have 94% sequence identity. CRABP I has a much wider tissue distribution in adults than CRABP II, which is most widely expressed during embryogenesis, and in human keratinocytes [10], [15], [16].
Several studies suggest that the function of CRABP I is to control the free concentration of RA and hence, its ability to interact with nuclear receptors. In the chick limb bud, for example, a CRABP I concentration gradient has been described which is inversely related to the total RA concentration [17]. In addition, when CRABP I is over-expressed in F9 stem cells, a higher concentration of RA is needed to initiate differentiation and vice versa [18]. Part of the biological function of CRABP may be in RA metabolism. RAin complex with CRABP I is a better substrate for metabolizing enzymes than freeRA [19]. CRABP I alters both the level and the type of metabolite produced [20].
A series of studies [21], [22], [23] has demonstrated a temporal and spatial distribution of CRBP and CRABP expression during embryogenesis which correlates well with the morphogenetic effects of the retinoids. These studies have led Chambon and co-workers to suggest that CRBP functions to store and release retinol in tissue where high levels of RA are required, whereas CRABP acts by sequestering RA in regions where normal development requires low RA levels. The ability of RA to induce the expression of the CRABP II gene suggests that CRABP II plays an important role in the regulation of skin growth and differentiation [10], [12].
Human CRABP I and II share 76% amino acid sequence identity. One would, therefore, expect their three-dimensional structures to be very similar with a Cα root mean square deviation (rmsd) of ∼0.6 Å [24]. On the basis of their amino acid sequences, both CRBP and CRABP could be placed in a family of cellular proteins that bind fatty acids and small lipids [25]. A more accurate alignment was made by Jones et al [26], who also suggested a structural basis for the sequence conservation observed within the family based on the structure determination of P2 myelin protein. The independent structure determinations of intestinal fatty acid binding protein (FABP) [27], [28]and P2 [26], [29] revealed a new protein fold, termed the β–clam by Banaszak and co-workers [27], in which the ligand is bound in a cavity between two orthogonal β–sheets built up from 10 strands. This fold shows some similarity with the retinol binding protein (RBP) fold, in which an eight-stranded β–barrel encloses the ligand [30], [31], [32].
The detailed interactions of intestinal FABP and P2 with their ligands were different. Now that more structures within this family have been solved, it appears that the interaction of the protein with the carboxylate of the fatty acid as observed in P2 is the most common mode of interaction [33]. A number of FABP structures have now been determined at medium and high resolution, both with and without bound ligands [29], [34], [35], [36], [37], [38], [39]. These proteins have similar structures and undergo only small changes upon binding ligands [37], [40]. The ligands adjust their own conformations to interact optimally with the internal hydrophobic surfaces of the protein [41]. In adipocyte lipid-binding protein, the tail of the ligand reaches to the surface [34].
The aligned sequences and the P2 structure enabled us to suggest that the trio of residues interacting with the carboxylate of the fatty acid may account for the observed specificity of CRBP and CRABP for their respective physiological ligands [26]. This has been confirmed by site-directed mutagenesis studies 42–44 and by the structure determination of CRBP I [29] and CRBP II [40]. However, although CRBP could be converted into an RA-binding protein [42], CRABP could not be converted into a retinol-binding protein [44].
We report herein the structures of bovine/murine CRABP I (identical amino acid sequences) and human CRABP II in complex with all-trans–RA (the natural ligand for CRABP I [45] and, until proven otherwise, the assumed natural ligand for CRABP II as well), and the structure of human CRABP II in complex with a synthetic retinoid, ‘compound 19’ (IUPAC name: 6-(2,3,4,5,6,7-hexahydro-2,4,4-trimethyl-1-methyl-eneind---yl)-3-methylhexa-2,4-dienoic acid) [46] Figure 1.
Section snippets
Results and discussion
Unless stated otherwise, we shall use the residue-numbering scheme of CRABP II without the amino-terminal methionine throughout this paper. Because of a deletion/insertion, residues 117–137 of CRABP II are structurally equivalent to residues 116–136 of CRABP I.
Biological implications
Vitamin A (retinol) and its active metabolite, retinoic acid (RA), exhibit profound effects on cell growth and differentiation, and on embryonic development. The biological activities of RA appear to be mediated by nuclear receptors which modulate the transcriptional activity of specific genes in a ligand–dependent fashion. Cellular RA–binding proteins (CRABPs) may function by controlling the free concentration of RA available to the nuclear receptors and hence, the ability of RA to interact
Protein preparation
The expression and purification of recombinant CRABP I was carried out as described previously [60]. CRABP II was expressed at high levels using the Escherichia coli strain BL21 DE3 [61] which carried the plasmid pET–3a/CRABP–II [12], [19]. The thawed cells were suspended in 50 mM bis–TRIS–propane/HCl (pH 7.0) containing 1 mM Na2 EDTA, 10% (w/v) saccharose, 0.05% (w/v) NaN3 , 2 mM 2–mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride and 0.25 mg of DNase per gram wet weight. The cells (10 ml of
Acknowledgements
This work was supported by Uppsala University and the Swedish Natural Science Research Council. We would like to thank Drs Alex Cameron and Jonas Uppenberg for processing the holo–CRABP II data, Dr Eleanor Dodson (University of York, UK) for her help with the use (and debugging) of the CCP4 version of AMoRe, and Dr. Axel Brünger (Yale University, New Haven, CT, USA) for his help with, and discussions about, the use of X–PLOR and the free R–factor.
Gerard Klaywegt, Terese Bergfors and T Alwyn Jones (corresponding author), Department of Molecular Biology, Uppsala University, Biomedical Center, Box 590, S–751 24 Uppsala, Sweden.
Hans Senn, Peter Le Motte and Bernard Gsell, Department of Pharmaceutical Research, F. Hoffmann–La Roche Ltd., CH–4002 Basel, Switzerland.
Koichi Shudo, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo–ku, Tokyo 113, Japan.
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Gerard Klaywegt, Terese Bergfors and T Alwyn Jones (corresponding author), Department of Molecular Biology, Uppsala University, Biomedical Center, Box 590, S–751 24 Uppsala, Sweden.
Hans Senn, Peter Le Motte and Bernard Gsell, Department of Pharmaceutical Research, F. Hoffmann–La Roche Ltd., CH–4002 Basel, Switzerland.
Koichi Shudo, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo–ku, Tokyo 113, Japan.