Prediction of Pesticide Interactions with Proteins Involved in Human Reproduction by Using a Virtual Screening Approach: A Case Study of Famoxadone Binding CRBP-III and Izumo.

In recent years, the awareness that pesticides can have other effects apart from generic toxicity is growing. In particular, several pieces of evidence highlight their influence on human fertility. In this study, we investigated, by a virtual screening approach, the binding between pesticides and proteins present in human gametes or associated with reproduction, in order to identify new interactions that could affect human fertility. To this aim, we prepared ligand (pesticides) and receptor (proteins) 3D structure datasets from online structural databases (such as PubChem and RCSB), and performed a virtual screening analysis using Autodock Vina. In the comparison of the predicted interactions, we found that famoxadone was predicted to bind Cellular Retinol Binding Protein-III in the retinol-binding site with a better minimum energy value of -10.4 Kcal/mol and an RMSD of 3.77 with respect to retinol (-7.1 Kcal/mol). In addition to a similar network of interactions, famoxadone binding is more stabilized by additional hydrophobic patches including L20, V29, A33, F57, L117, and L118 amino acid residues and hydrogen bonds with Y19 and K40. These results support a possible competitive effect of famoxadone on retinol binding with impacts on the ability of developing the cardiac tissue, in accordance with the literature data on zebrafish embryos. Moreover, famoxadone binds, with a minimum energy value between -8.3 and -8.0 Kcal/mol, to the IZUMO Sperm-Egg Fusion Protein, interacting with a network of polar and hydrophobic amino acid residues in the cavity between the 4HB and Ig-like domains. This binding is more stabilized by a predicted hydrogen bond with the N185 residue of the protein. A hindrance in this position can probably affect the conformational change for JUNO binding, avoiding the gamete membrane fusion to form the zygote. This work opens new interesting perspectives of study on the effects of pesticides on fertility, extending the knowledge to other typologies of interaction which can affect different steps of the reproductive process.

Retinol-binding protein 2 (RBP2): biology and pathobiology.

Retinol-binding protein 2 (RBP2; originally cellular retinol-binding protein, type II (CRBPII)) is a 16 kDa cytosolic protein that in the adult is localized predominantly to absorptive cells of the proximal small intestine. It is well established that RBP2 plays a central role in facilitating uptake of dietary retinoid, retinoid metabolism in enterocytes, and retinoid actions locally within the intestine. Studies of mice lacking Rbp2 establish that Rbp2 is not required in times of dietary retinoid-sufficiency. However, in times of dietary retinoid-insufficiency, the complete lack of Rbp2 gives rise to perinatal lethality owing to RBP2 absence in both placental (maternal) and neonatal tissues. Moreover, when maintained on a high-fat diet, Rbp2-knockout mice develop obesity, glucose intolerance and a fatty liver. Unexpectedly, recent investigations have demonstrated that RBP2 binds long-chain 2-monoacylglycerols (2-MAGs), including the canonical endocannabinoid 2-arachidonoylglycerol, with very high affinity, equivalent to that of retinol binding. Crystallographic studies establish that 2-MAGs bind to a site within RBP2 that fully overlaps with the retinol binding site. When challenged orally with fat, mucosal levels of 2-MAGs in Rbp2 null mice are significantly greater than those of matched controls establishing that RBP2 is a physiologically relevant MAG-binding protein. The rise in MAG levels is accompanied by elevations in circulating levels of the hormone glucose-dependent insulinotropic polypeptide (GIP). It is not understood how retinoid and/or MAG binding to RBP2 affects the functions of this protein, nor is it presently understood how these contribute to the metabolic and hormonal phenotypes observed for Rbp2-deficient mice.

Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation.

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid-binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the kcat/Km values either decreased 5-fold or were equal to the respective free retinoid values. The kcat/Km value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in kcat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.

Organic Solvents for Enhanced Proteolysis of Stable Proteins for Hydrogen-Deuterium Exchange Mass Spectrometry.

Protein digestion is a key challenge in mass spectrometry (MS)-based structural proteomics. Although using hydrogen-deuterium exchange kinetics with MS (HDX-MS) to interrogate the high-order structure of proteins is now established, it can be challenging for ß-barrel proteins, which are important in cellular transport. These proteins contain a continuous chain of H-bonds that impart stability, causing difficulty in digestion for bottom-up measurements. To overcome this impediment, we tested organic solvents as denaturants during on-line pepsin digestion of soluble ß-barrel proteins. We selected green fluorescent protein (GFP), siderocalin (Scn), and retinol-binding protein 4 (RBP4) as model proteins and screened six different polar-aprotic and polar-protic solvent combinations to disrupt the H-bonds and hydrophobic interactions holding together the ß-sheets. The use of organic solvents improves digestion, generating more peptides from the rigid ß-barrel regions, without compromising the ability to predict the retinol binding site on RBP4 when adopting this proteolysis with HDX.

Human Cellular Retinol Binding Protein II Forms a Domain-Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering.

Domain-swapping is a mechanism for evolving new protein structure from extant scaffolds, and has been an efficient protein-engineering strategy for tailoring functional diversity. However, domain swapping can only be exploited if it can be controlled, especially in cases where various folds can coexist. Herein, we describe the structure of a domain-swapped trimer of the iLBP family member hCRBPII, and suggest a mechanism for domain-swapped trimerization. It is further shown that domain-swapped trimerization can be favored by strategic installation of a disulfide bond, thus demonstrating a strategy for fold control. We further show the domain-swapped trimer to be a useful protein design template by installing a high-affinity metal binding site through the introduction of a single mutation, taking advantage of its threefold symmetry. Together, these studies show how nature can promote oligomerization, stabilize a specific oligomer, and generate new function with minimal changes to the protein sequence.

Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging.

A reengineered human cellular retinol binding protein II (hCRBPII), a 15-kDa protein belonging to the intracellular lipid binding protein (iLBP) family, generates a highly fluorescent red pigment through the covalent linkage of a merocyanine aldehyde to an active site lysine residue. The complex exhibits "turn-on" fluorescence, due to a weakly fluorescent aldehyde that "lights up" with subsequent formation of a strongly fluorescent merocyanine dye within the binding pocket of the protein. Cellular penetration of merocyanine is rapid, and fluorophore maturation is nearly instantaneous. The hCRBPII/merocyanine complex displays high quantum yield, low cytotoxicity, specificity in labeling organelles, and compatibility in both cancer cell lines and yeast cells. The hCRBPII/merocyanine tag is brighter than most common red fluorescent proteins.

Identification of RBP4 from bighead carp (Hypophthalmichthys nobilis) / silver carp (Hypophthalmichthys molitrix) and effects of CpG ODN on RBP4 expression under A. hydrophila challenge.

Retinol-binding protein 4 (RBP4) is known as a highly conserved adipokine for immune activation. Aeromonas hydrophila (A. hydrophila) is the most common zoonotic pathogen in aquaculture, which causes serious economic losses to aquaculture, especially to bighead carp (Hypophthalmichthys nobilis, H. nobilis) and silver carp (Hypophthalmichthys molitrix, H. molitrix). Recent studies along with our previous findings have shown that synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) can play a good role in aquatic animals against infection. In order to clarify the relationship between CpG ODN and RBP4 under A. hydrophila infection, firstly, full-length RBP4 cDNAs from H. nobilis and H. molitrix were cloned. And characteristics of RBP4, including sequence and structure, tissue distribution and genetic evolution were analyzed. In addition, mRNA expression levels of RBP4, cytokine, toll-like receptors (TLRs), morbidity and survival rates of H. nobilis and H. molitrix were observed post CpG ODN immunization or following challenge. The results indicated that hn/hm_RBP4 (RBP4 genes obtained from H. nobilis and H. molitrix) had the highest homology with Megalobrama amblycephala. Distribution data showed that the expression level of hn_RBP4 mRNA was higher than that of hm_RBP4. After CpG ODN immunization followed by A.hydrophila challenge, significantly higher survival was observed in both carps, together with up-regulated RBP4 expression. Meanwhile, hn/hm_IL-1ß level was relatively flat (and decreased), hn/hm_IFN-γ, hn/hm_TLR4 and hn/hm_TLR9 levels increased significantly, but hn/hm_STRA6 showed no significant change, compared with control. Moreover, CpG ODN immunization could induce stronger immune protective responses (higher IFN-γ/gentle IL-1ß level and lower morbidity/higher survival rate) against A. hydrophila in H. nobilis, along with higher RBP4 level, when compared with that in H. molitrix. These results demonstrated that RBP4 was well involved in the immune protection of CpG ODN. Based on the results, we speculated that in the case of A. hydrophila infection, TLR9 signaling pathway was activated by CpG ODN. Subsequently, CpG ODN up-regulated RBP4, and RBP4 activated TLR4 signaling pathway. Then TLR4 and TLR9 synergistically improved the anti-infection responses. Our findings have good significance for improving resistance to pathogen infection in freshwater fish.

Engineering the hCRBPII Domain-Swapped Dimer into a New Class of Protein Switches.

Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has proven challenging, with only a few known examples that are not derivatives of naturally occurring allosteric systems. We have discovered that the domain-swapped (DS) dimer of hCRBPII undergoes a large and robust conformational change upon retinal binding, making it a potentially powerful template for the design of protein conformational switches. Atomic resolution structures of the apo- and holo-forms illuminate a simple, mechanical movement involving sterically driven torsion angle flipping of two residues that drive the motion. We further demonstrate that the conformational "readout" can be altered by addition of cross-domain disulfide bonds, also visualized at atomic resolution. Finally, as a proof of principle, we have created an allosteric metal binding site in the DS dimer, where ligand binding results in a reversible 5-fold loss of metal binding affinity. The high resolution structure of the metal-bound variant illustrates a well-formed metal binding site at the interface of the two domains of the DS dimer and confirms the design strategy for allosteric regulation.

Cannabinoid Interactions with Proteins: Insights from Structural Studies.

Cannabinoids have been widely used for recreational and medicinal purposes. The increasing legalization of cannabinoid use and the growing success in Medicinal Chemistry of cannabinoids have fueled recent interest in cannabinoid-sensing sites in receptor proteins. Here, we review structural data from high-resolution cryo-EM and crystallography studies that depict phytocannabinoid, endocannabinoid, and synthetic cannabinoid molecules bound to various proteins. The latter include antigen-binding fragment (Fab), cellular retinol binding protein 2 (CRBP2), fatty acid-binding protein 5 (FABP5), peroxisome proliferator-activated receptor γ (PPAR γ), and cannabinoid receptor types 1 and 2 (CB1 and CB2). Cannabinoid-protein complexes reveal the complex design of cannabinoid binding sites that are usually presented by conventional ligand-binding pockets on respective proteins. However, subtle differences in cannabinoid interaction with amino acids within the binding pocket often result in diverse consequences for protein function. The rapid increase in available structural data on cannabinoid-protein interactions will ultimately direct drug design efforts toward rendering highly potent cannabinoid-related pharmacotherapies that are devoid of side effects.

A Genetically Encoded Ratiometric pH Probe: Wavelength Regulation-Inspired Design of pH Indicators.

Mutants of human cellular retinol-binding protein II (hCRBPII) were engineered to bind a julolidine retinal analogue for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, which affects the absorption and, thus, the emission of the protein/fluorophore complex. The ratio of emissions obtained at two excitation wavelengths that correspond to the absorption of the two forms of the protein/fluorophore complex, leads to a concentration-independent measure of pH.

Deciphering protein dynamics changes along the pathway of retinol uptake by cellular retinol-binding proteins 1 and 2.

Four Cellular Retinol-binding Proteins (CRBP 1, 2, 3, 4) are encoded in the human genome. CRBP 1 and 2, sharing a 56% amino acid sequence identity, exhibit the highest binding affinities for retinol. Previous NMR studies provided some insights into the mechanism of retinol uptake, but details of such mechanism remain to be elucidated. Herein, the results of molecular dynamics simulations for the uptake of retinol by CRBP 1 and 2 are consistent with the presence of two different retinol entry points, both involving the 'cap region' (α-helices I and II and neighboring loops). We observed that a hydrophobic patch at the surface of the 'portal region' (α-helix II, CD and EF loops) of CRBP 1 attracts retinol, which accesses the binding cavity through an opening generated by the concerted movements of Arg58 and Phe57, present in the CD loop. In CRBP 2 a different distribution of the surface residues of the 'cap region' allows retinol to access the binding cavity by sinking in a hydrophobic matrix between the two α-helices. Polar interactions mainly affect retinol movements inside the ß-barrel cavities of both CRBPs. The interaction energy profiles are in agreement with the different behavior of the two protein systems.

Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between ß-strands ßC-ßD and ßE-ßF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins.

Computing the binding affinity of a ligand buried deep inside a protein with the hybrid steered molecular dynamics.

Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions. These errors propagate to the final results of the computed binding affinities even if we were able to perfectly implement the statistical mechanics of all the processes relevant to a given problem. And they are actually amplified to various degrees even in the mature, sophisticated computational approaches. In particular, the free energy perturbation (alchemical) approaches amplify the errors in the force field parameters because they rely on extracting the small differences between similarly large numbers. In this paper, we develop a hybrid steered molecular dynamics (hSMD) approach to the difficult binding problems of a ligand buried deep inside a protein. Sampling the transition along a physical (not alchemical) dissociation path of opening up the binding cavity---pulling out the ligand---closing back the cavity, we can avoid the problem of error amplifications by not relying on small differences between similar numbers. We tested this new form of hSMD on retinol inside cellular retinol-binding protein 1 and three cases of a ligand (a benzylacetate, a 2-nitrothiophene, and a benzene) inside a T4 lysozyme L99A/M102Q(H) double mutant. In all cases, we obtained binding free energies in close agreement with the experimentally measured values. This indicates that the force field parameters we employed are accurate and that hSMD (a brute force, unsophisticated approach) is free from the problem of error amplification suffered by many sophisticated approaches in the literature.

Functions of Intracellular Retinoid Binding-Proteins.

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Tuning the Protein-Induced Absorption Shifts of Retinal in Engineered Rhodopsin Mimics.

Rational design of light-capturing properties requires understanding the molecular and electronic structure of chromophores in their native chemical or biological environment. We employ here large-scale quantum chemical calculations to study the light-capturing properties of retinal in recently designed human cellular retinol binding protein II (hCRBPII) variants (Wang et al. Science, 2012, 338, 1340-1343). Our calculations show that these proteins absorb across a large part of the visible spectrum by combined polarization and electrostatic effects. These effects stabilize the ground or excited state energy levels of the retinal by perturbing the Schiff-base or ß-ionone moieties of the chromophore, which in turn modulates the amount of charge transfer within the molecule. Based on the predicted tuning principles, we design putative in silico mutations that further shift the absorption properties of retinal in hCRBPII towards the ultraviolet and infrared regions of the spectrum.

Molecular Mechanism of Wide Photoabsorption Spectral Shifts of Color Variants of Human Cellular Retinol Binding Protein II.

Color variants of human cellular retinol binding protein II (hCRBPII) created by protein engineering were recently shown to exhibit anomalously wide photoabsorption spectral shifts over ∼200 nm across the visible region. The remarkable phenomenon provides a unique opportunity to gain insight into the molecular basis of the color tuning of retinal binding proteins for understanding of color vision as well as for engineering of novel color variants of retinal binding photoreceptor proteins employed in optogenetics. Here, we report a theoretical investigation of the molecular mechanism underlying the anomalously wide spectral shifts of the color variants of hCRBPII. Computational modeling of the color variants with hybrid molecular simulations of free energy geometry optimization succeeded in reproducing the experimentally observed wide spectral shifts, and revealed that protein flexibility, through which the active site structure of the protein and bound water molecules is altered by remote mutations, plays a significant role in inducing the large spectral shifts.

Structures of holo wild-type human cellular retinol-binding protein II (hCRBPII) bound to retinol and retinal.

Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.

Regulation of Absorption and Emission in a Protein/Fluorophore Complex.

Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore's aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission. The protein is readily expressed in mammalian systems and binds with exogenously delivered fluorophore as demonstrated by live-cell imaging experiments.

Retinol-binding protein 2 (RBP2): More than just dietary retinoid uptake.

Retinol-binding protein 2 (RBP2, also known as cellular retinol-binding protein 2 (CRBP2)) is a member of the fatty acid-binding protein family and has been extensively studied for its role in facilitating dietary vitamin A (retinol) uptake and metabolism within enterocytes of the small intestine. RBP2 is present in highest concentrations in the proximal small intestine where it constitutes approximately 0.1-0.5% of soluble protein. Recent reports have established that RBP2 binds monoacylglycerols (MAGs) with high affinity, including the canonical endocannabinoid 2-arachidonoylglycerol (2-AG). Crystallographic studies reveal that retinol, 2-AG, or other long-chain MAGs alternatively can bind in the retinol-binding pocket of RBP2. It also has been demonstrated recently that Rbp2-deficient mice are more susceptible to developing obesity and associated metabolic phenotypes when exposed to a high fat diet, or as they age when fed a conventional chow diet. When subjected to an oral fat challenge, the Rbp2-deficient mice release into the circulation significantly more, compared to littermate controls, of the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP). These new findings regarding RBP2 structure and actions within the intestine are the focus of this review.

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers.

The main retinol carriers in the cytosol are the cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II), which exhibit distinct tissue distributions. They play different roles in the maintenance of vitamin A homeostasis and feature a 100-fold difference in retinol affinity whose origin has not been described in detail. NMR-based hydrogen/deuterium exchange measurements show that, while retinol binding endows both proteins with a more rigid structure, many amide protons exchange much faster in CRBP-II than in CRBP-I in both apo and holo form, despite the conserved three-dimensional fold. The remarkable difference in intrinsic stability between the two homologs appears to modulate their binding properties: the stronger retinol binder CRBP-I displays a reduced flexibility of the backbone structure with respect to CRBP-II. This difference must derive from specific evolution-based amino acid substitutions, resulting in additional stabilization of the CRBP-I scaffold: in fact, we identified a number of potential salt bridges on the protein surface as well as several key interactions inside the binding cavity. Furthermore, our NMR data demonstrate that helix alphaII of the characteristic helix-turn-helix motif in the ligand portal region exists in both apo and holo CRBP-II. Hence, the previously proposed model of retinol binding needs to be revised.