3-O-Methyltolcapone and Its Lipophilic Analogues Are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity.

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine in blood and cerebrospinal fluid. To date, more than 130 TTR point mutations are known to destabilise the TTR tetramer, leading to its extracellular pathological aggregation accumulating in several organs, such as heart, peripheral and autonomic nerves, and leptomeninges. Tolcapone is an FDA-approved drug for Parkinson's disease that has been repurposed as a TTR stabiliser. We characterised 3-O-methyltolcapone and two newly synthesized lipophilic analogues, which are expected to be protected from the metabolic glucuronidation that is responsible for the lability of tolcapone in the organism. Immunoblotting assays indicated the high degree of TTR stabilisation, coupled with binding selectivity towards TTR in diluted plasma of 3-O-methyltolcapone and its lipophilic analogues. Furthermore, in vitro toxicity data showed their several-fold improved neuronal and hepatic safety compared to tolcapone. Calorimetric and structural data showed that both T4 binding sites of TTR are occupied by 3-O-methyltolcapone and its lipophilic analogs, consistent with an effective TTR tetramer stabilisation. Moreover, in vitro permeability studies showed that the three compounds can effectively cross the blood-brain barrier, which is a prerequisite for the inhibition of TTR amyloidogenesis in the cerebrospinal fluid. Our data demonstrate the relevance of 3-O-methyltolcapone and its lipophilic analogs as potent inhibitors of TTR amyloidogenesis.

Interactions of tolcapone analogues as stabilizers of the amyloidogenic protein transthyretin.

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine and retinol in blood and cerebrospinal fluid. TTR stabilizers, such as tolcapone, an FDA approved drug for Parkinson's disease, are able to interact with residues of the thyroxine-binding sites of TTR, both wild type and pathogenic mutant forms, thereby stabilizing its tetrameric native state and inhibiting amyloidogenesis. Herein, we report on the synthesis of 3-deoxytolcapone, a novel stabilizer of TTR. The high-resolution X-ray analyses of the interactions of 3-O-methyltolcapone and 3-deoxytolcapone with TTR were performed. In the two TTR-ligand complexes the tolcapone analogues establish mainly H-bond and hydrophobic interactions with residues of the thyroxine-binding site of the TTR tetramer. Both compounds are capable of high and selective stabilization of TTR in the presence of plasma proteins, despite their markedly different 'forward' and 'reverse' binding mode, respectively. In fact, the loss or the weakening of stabilizing interactions with protein residues of 3-deoxytolcapone in comparison with tolcapone and 3-O-methyltolcapone is compensated by new interactions established at the dimer-dimer interface. Our data, coupled with previously reported data on the pharmacokinetics properties in humans of tolcapone and 3-O-methyltolcapone, further support the relevance of the latter tolcapone analogue as TTR stabilizer.

Structure-activity relationships of flurbiprofen analogues as stabilizers of the amyloidogenic protein transthyretin.

The inherent amyloidogenic potentialof wild type transthyretin (TTR) is enhanced by a large number of point mutations, which destabilize the TTR tetramer, thereby promoting its disassembly and pathological aggregation responsible for TTR-related amyloidosis. TTR stabilizers are able to interact with the thyroxine-binding sites of TTR, stabilizing its tetrameric native state and inhibiting amyloidogenesis. Herein, we report on in vitro, ex vivo, and X-ray analyses to assess the TTR structural stabilization by analogues of flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID). Overall, considering together binding selectivity and protective effects on TTR native structure by flurbiprofen analogues in the presence of plasma proteins, as determined by Western Blot,the aforementioned properties of analyzed compounds appear to be better (CHF5075 and CHF4802) or similar (CHF4795) or worse (CHF5074, also known as CSP-1103) as compared to those of diflunisal, used as a reference TTR stabilizer. Molecular details of the determinants affecting the interactionsof CHF5075, CHF4802, and CHF4795 with wild type TTRand of CHF5074 withtheamyloidogenic A25TTTR variant havebeen elucidated by X-ray analysis. Distinct interactions with TTR appear to characterize flurbiprofen analogues and the NSAID diflunisal and its analogues as TTR stabilizers. Relationships between stabilizing effect on TTR by flurbiprofen analogues determined experimentally and molecular details of their interactions with TTR have been established, providing the rationale for their protective effects on the native protein structure.

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.

Structural and molecular determinants affecting the interaction of retinol with human CRBP1.

Four cellular retinol-binding protein (CRBP) types (CRBP1,2,3,4) are encoded in the human genome. Here, we report on X-ray analyses of human apo- and holo-CRBP1, showing nearly identical structures, at variance with the results of a recent study on the same proteins containing a His-Tag, which appears to be responsible for a destabilizing effect on the apoprotein. The analysis of crystallographic B-factors for our structures indicates that the putative portal region, in particular α-helix-II, along with Arg58 and the E-F loop, is the most flexible part of both apo- and holoprotein, consistent with its role in ligand uptake and release. Fluorometric titrations of wild type and mutant forms of apo-CRBP1, coupled with X-ray analyses, provided insight into structural and molecular determinants for the interaction of retinol with CRBP1. An approximately stoichiometric binding of retinol to wild type apo-CRBP1 (Kd∼4.5nM), significantly lower binding affinity for both mutants Q108L (Kd∼65nM) and K40L (Kd∼70nM) and very low binding affinity for the double mutant Q108L/K40L (Kd∼250nM) were determined, respectively. Overall, our data indicate that the extensive apolar interactions between the ligand and hydrophobic residues lining the retinol binding cavity are sufficient to keep it in its position bound to CRBP1. However, polar interactions of the retinol hydroxyl end group with Gln108 and Lys40 play a key role to induce a high binding affinity and specificity for the interaction.

Transthyretin Binding Heterogeneity and Anti-amyloidogenic Activity of Natural Polyphenols and Their Metabolites.

Transthyretin (TTR) is an amyloidogenic protein, the amyloidogenic potential of which is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of x-ray analysis of TTR in complex with both ligands. Although revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, whereas genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulfate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by x-ray analysis of their complexes with TTR.

Structural evidence for asymmetric ligand binding to transthyretin.

Human transthyretin (TTR) represents a notable example of an amyloidogenic protein, and several compounds that are able to stabilize its native state have been proposed as effective drugs in the therapy of TTR amyloidosis. The two thyroxine (T4) binding sites present in the TTR tetramer display negative binding cooperativity. Here, structures of TTR in complex with three natural polyphenols (pterostilbene, quercetin and apigenin) have been determined, in which this asymmetry manifests itself as the presence of a main binding site with clear ligand occupancy and related electron density and a second minor site with a much lower ligand occupancy. The results of an analysis of the structural differences between the two binding sites are consistent with such a binding asymmetry. The different ability of TTR ligands to saturate the two T4 binding sites of the tetrameric protein can be ascribed to the different affinity of ligands for the weaker binding site. In comparison, the high-affinity ligand tafamidis, co-crystallized under the same experimental conditions, was able to fully saturate the two T4 binding sites. This asymmetry is characterized by the presence of small but significant differences in the conformation of the cavity of the two binding sites. Molecular-dynamics simulations suggest the presence of even larger differences in solution. Competition binding assays carried out in solution revealed the presence of a preferential binding site in TTR for the polyphenols pterostilbene and quercetin that was different from the preferential binding site for T4. The TTR binding asymmetry could possibly be exploited for the therapy of TTR amyloidosis by using a cocktail of two drugs, each of which exhibits preferential binding for a distinct binding site, thus favouring saturation of the tetrameric protein and consequently its stabilization.

First trimester concentrations of the TTR-RBP4-retinol complex components as early markers of insulin-treated gestational diabetes mellitus.

BACKGROUND: The objective of the study was to investigate the relationship between first trimester maternal serum levels of the TTR-RBP4-ROH complex components and the later insurgence of an altered glucose metabolism during pregnancy. METHODS: Retrospective case control study including 96 patients between the 12th and 14th week of gestation, 32 that developed gestational diabetes mellitus (GDM), respectively, 21 non-insulin-treated (dGDM) and 11 insulin-treated (iGDM), 20 large for gestational age fetuses (LGA) without GDM and 44 patients with normal outcome as control. Serum concentrations of RBP4 and TTR were assessed by ELISA; serum concentration of ROH by reverse-phase high performance liquid chromatography (rpHPLC). The molecular heterogeneity of TTR and RBP4 was analyzed after immunoprecipitation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: iGDM patients were characterized by reduced TTR, RBP4 and ROH compared to controls (respectively, iGDM vs. controls, mean±SD: TTR 3.96±0.89 µmol/L vs. 4.68±1.21 µmol/L, RBP4 1.13±0.25 µmol/L vs. 1.33±0.38 µmol/L and ROH 1.33±0.17 µmol/L vs. 1.62±0.29 µmol/L, p<0.05). TTR containing Gly10 in place of Cys10 was lower in the iGDM group (p<0.05) compared to controls. In the final logistic regression model ROH significantly predicted the diagnosis of iGDM (OR 0.93, 95% CI 0.87-0.98, p<0.05). CONCLUSIONS: First trimester maternal serum ROH, RBP4 and TTR represent potential biomarkers associated with the development of iGDM.

An aminotransferase branch point connects purine catabolism to amino acid recycling.

Although amino acids are known precursors of purines, a pathway for the direct recycling of amino acids from purines has never been described at the molecular level. We provide NMR and crystallographic evidence that the PucG protein from Bacillus subtilis catalyzes the transamination between an unstable intermediate ((S)-ureidoglycine) and the end product of purine catabolism (glyoxylate) to yield oxalurate and glycine. This activity enables soil and gut bacteria to use the animal purine waste as a source of carbon and nitrogen. The reaction catalyzed by (S)-ureidoglycine-glyoxylate aminotransferase (UGXT) illustrates a transamination sequence in which the same substrate provides both the amino group donor and, via its spontaneous decay, the amino group acceptor. Structural comparison and mutational analysis suggest a molecular rationale for the functional divergence between UGXT and peroxisomal alanine-glyoxylate aminotransferase, a fundamental enzyme for glyoxylate detoxification in humans.

Structural characterization of recombinant crustacyanin subunits from the lobster Homarus americanus.

Crustacean crustacyanin proteins are linked to the production and modification of carapace colour, with direct implications for fitness and survival. Here, the structural and functional properties of the two recombinant crustacyanin subunits H(1) and H(2) from the American lobster Homarus americanus are reported. The two subunits are structurally highly similar to the corresponding natural apo crustacyanin CRTC and CRTA subunits from the European lobster H. gammarus. Reconstitution studies of the recombinant crustacyanin proteins H(1) and H(2) with astaxanthin reproduced the bathochromic shift of 85-95 nm typical of the natural crustacyanin subunits from H. gammarus in complex with astaxanthin. Moreover, correlations between the presence of crustacyanin genes in crustacean species and the resulting carapace colours with the spectral properties of the subunits in complex with astaxanthin confirmed this genotype-phenotype linkage.

Amyloidogenic potential of transthyretin variants: insights from structural and computational analyses.

Human transthyretin (TTR) is an amyloidogenic protein whose mild amyloidogenicity is enhanced by many point mutations affecting considerably the amyloid disease phenotype. To ascertain whether the high amyloidogenic potential of TTR variants may be explained on the basis of the conformational change hypothesis, an aim of this work was to determine structural alterations for five amyloidogenic TTR variants crystallized under native and/or destabilizing (moderately acidic pH) conditions. While at acidic pH structural changes may be more significant because of a higher local protein flexibility, only limited alterations, possibly representing early events associated with protein destabilization, are generally induced by mutations. This study was also aimed at establishing to what extent wild-type TTR and its amyloidogenic variants are intrinsically prone to beta-aggregation. We report the results of a computational analysis predicting that wild-type TTR possesses a very high intrinsic beta-aggregation propensity which is on average not enhanced by amyloidogenic mutations. However, when located in beta-strands, most of these mutations are predicted to destabilize the native beta-structure. The analysis also shows that rat and murine TTR have a lower intrinsic beta-aggregation propensity and a similar native beta-structure stability compared with human TTR. This result is consistent with the lack of in vitro amyloidogenicity found for both murine and rat TTR. Collectively, the results of this study support the notion that the high amyloidogenic potential of human pathogenic TTR variants is determined by the destabilization of their native structures, rather than by a higher intrinsic beta-aggregation propensity.

Acidic pH-induced conformational changes in amyloidogenic mutant transthyretin.

Several proteins, including transthyretin (TTR), can generate in tissues extracellular insoluble aggregates, in the form of fibrils, that are associated with pathological states known as amyloidoses. To date, more than 80 different TTR point mutations have been associated with hereditary amyloidosis in humans. In vitro, the formation of amyloid fibrils by human TTR is known to be triggered by acidic pH. We show here that, in vitro, the natural amyloidogenic I84S and the non-natural I84A TTR mutant forms exhibit a propensity to produce fibrils in an acidic medium significantly higher than that of wild-type TTR. The two mutant forms have been crystallized at both neutral and acidic pH. Their neutral pH crystal structures are very similar to that of wild-type TTR, consistent with previous evidence indicating that only minor structural changes are induced by amyloidogenic mutations. On the contrary, their crystal structures at moderately low pH (4.6) show significant conformational differences as compared to their neutral pH structures. Remarkably, such changes are not induced in wild-type TTR crystallized at low pH. The most relevant consist of the unwinding of the TTR short alpha-helix and of the change in conformation of the loop connecting the alpha-helix to beta-strand F. Only one monomer of the crystallographic dimer is affected, causing a disruption of the tetrameric symmetry. This asymmetry and a possible destabilization of the tetrameric quaternary structure of TTR may be responsible for the amyloidogenic potential of the two TTR mutant forms at low pH.

Cloning, E. coli overexpression, purification and binding properties of TraA and TraC, two proteins involved in the pheromone-dependent conjugation process in enterococci.

The bacteriocin encoding plasmid pPD1 from Enterococcus faecalis is involved in a mating response to the sex pheromone cPD1 produced by recipient bacterial cells devoid of pPD1. Previous studies showed that cPD1 is internalized into donor cells in a process in which TraC plays the role of cell surface pheromone receptor. Inside the recipient cells, the pheromone binds to the plasmid-encoded cytoplasmic protein TraA, able to recognize specific DNA sequences and to modulate the conjugation process. To avoid self-induction of the conjugation process, donor cells produce the inhibitor iPD1, which competes with cPD1. This study was designed to produce recombinant TraA and TraC in a functionally active state and to evaluate their main functional properties. We have isolated the sequences encoding TraA and TraC from the plasmid pPD1 and cloned them in suitable expression vectors. The two recombinant proteins were successfully obtained in a soluble form using Escherichia coli as expression host and a T7 inducible expression system. TraC and TraA were purified to homogeneity by three or two chromatographic steps, respectively, leading to a final yield up to 4mg/l of cell culture for TraC and up to 10mg/l of cell culture for TraA. The ability of TraA and TraC to bind the specific pheromone and inhibitor peptides has been assessed by means of ESI-mass spectrometry. Moreover, the ability of recombinant TraA to bind DNA has been demonstrated by means of electrophoretic mobility shift assay. Overall these results are consistent with the heterologously expressed TraC and TraA being functionally active.

Dynamics and Thermodynamics of Transthyretin Association from Molecular Dynamics Simulations.

Molecular dynamics simulations are used in this work to probe the structural stability and the dynamics of engineered mutants of transthyretin (TTR), i.e., the double mutant F87M/L110M (MT-TTR) and the triple mutant F87M/L110M/S117E (3M-TTR), in relation to wild-type. Free energy analysis from end-point simulations and statistical effective energy functions are used to analyze trajectories, revealing that mutations do not have major impact on protein structure but rather on protein association, shifting the equilibria towards dissociated species. The result is confirmed by the analysis of 3M-TTR which shows dissociation within the first 10 ns of the simulation, indicating that contacts are lost at the dimer-dimer interface, whereas dimers (formed by monomers which pair to form two extended ß-sheets) appear fairly stable. Overall the simulations provide a detailed view of the dynamics and thermodynamics of wild-type and mutant transthyretins and a rationale of the observed effects.

Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter.

During early vertebrate evolution, a duplication event in the gene encoding 5-hydroxyisourate hydrolase (HIUase), a widely distributed enzyme of purine metabolism, gave rise to transthyretin (TTR), a thyroid hormone transporter. We report here on the crystal structure of zebra fish HIUase in two different crystal forms. Despite the phylogenetic distance, this structure compares well with those of newly characterized bacterial HIUases, especially with regard to catalytic regions, which are highly preserved. Comparison with TTR structure reveals a highly conserved scaffold, harbouring distinct functional sites located in the same regions of the two vertebrate proteins. Residues that are differentially conserved in HIUases compared to TTR map in putative catalytic regions occupying significant portions of the two halves of a central channel that transverses the whole TTR protein. The evolution of TTR has been accompanied by remarkable changes of the HIUase active sites that gave rise to a channel open at both ends, thus allowing free access to hormone molecules.

Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens.

This study describes the three-dimensional crystal structure of a non-specific lipid transport protein (ns-LTP) from Rosaceae. Whilst ns-LTPs from species other than Rosaceae, such as nuts, cereals, grape, oranges and vegetables are also responsible for plant food allergies, this is less frequent compared with ns-LTPs from Rosaceae in the Mediterranean area. In this heterologously expressed peach Pru p3, a ligand is present inside the central cavity of the protein, presumably a fatty acid that was present or produced in the culture medium of the expression organism Escherichia coli. Moreover, the two molecules of ns-LTP present in the asymmetric unit bind this ligand in a different way, suggesting a significant degree of plasticity for the peach ns-LTP binding cavity, despite the presence of four disulphide bridges. Two molecules are present in the asymmetric unit: molecule A is a fully liganded protein, while molecule B apparently represents a partially liganded state. Also, molecular dynamics simulation, along with other evidence, suggests that these two molecular conformations represent different states in solution. Comparison of the 3D models of different ns-LTPs justifies the evidence of a high degree of conservation of the putative IgE binding epitopes among proteins of the Rosaceae family and the presence of significant amino acid replacements in correspondence of the same regions in ns-LTPs of botanical species unrelated to Rosaceae.

Structural and dynamics evidence for scaffold asymmetric flexibility of the human transthyretin tetramer.

The molecular symmetry of multimeric proteins is generally determined by using X-ray diffraction techniques, so that the basic question as to whether this symmetry is perfectly preserved for the same protein in solution remains open. In this work, human transthyretin (TTR), a homotetrameric plasma transport protein with two binding sites for the thyroid hormone thyroxine (T4), is considered as a case study. Based on the crystal structure of the TTR tetramer, a hypothetical D2 symmetry is inferred for the protein in solution, whose functional behavior reveals the presence of two markedly different Kd values for the two T4 binding sites. The latter property has been ascribed to an as yet uncharacterized negative binding cooperativity. A triple mutant form of human TTR (F87M/L110M/S117E TTR), which is monomeric in solution, crystallizes as a tetrameric protein and its structure has been determined. The exam of this and several other crystal forms of human TTR suggests that the TTR scaffold possesses a significant structural flexibility. In addition, TTR tetramer dynamics simulated using normal modes analysis exposes asymmetric vibrational patterns on both dimers and thermal fluctuations reveal small differences in size and flexibility for ligand cavities at each dimer-dimer interface. Such small structural differences between monomers can lead to significant functional differences on the TTR tetramer dynamics, a feature that may explain the functional heterogeneity of the T4 binding sites, which is partially overshadowed by the crystal state.

Ligand-binding specificity of an invertebrate (Manduca sexta) putative cellular retinoic acid binding protein.

Intracellular lipid-binding proteins (iLBPs) are small cytoplasmic proteins that specifically interact with hydrophobic ligands. Fatty acid-binding proteins (FABPs), cellular retinoic acid-binding proteins (CRABPs) and cellular retinol-binding proteins (CRBPs) belong to the iLBP family. A recently identified insect (Manduca sexta) iLBP has been reported to possibly represent an invertebrate CRABP mimicking the role of CRABPs in vertebrate organisms. The presence in this protein of the characteristic binding triad residues involved in the interaction with ligand carboxylate head groups, a feature pertaining to several FABPs and to CRABPs, and the close phylogenetic relationships with both groups of vertebrate heart-type FABPs and CRBPs/CRABPs, makes it difficult to assign it to either FABPs or CRABPs. However, its negligible interaction with retinoic acid and high affinity (K(d) values in the 10(-8) M range) for fatty acids have been established by means of direct and competitive binding assays. As shown by phylogenetic analysis, the M. sexta iLBP belongs to a wide group of invertebrate iLBPs, which, besides being closely related phylogenetically, share distinctive features, such as the conservation of chemically distinct residues in their amino acid sequences and the ability to bind fatty acids. Our results are in keeping with the lack of cellular retinoid-binding proteins in invertebrates and with their later appearance during the course of chordate evolution.

Catalysis and Structure of Zebrafish Urate Oxidase Provide Insights into the Origin of Hyperuricemia in Hominoids.

Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaelis constant, possibly near to upper end of the normal range of urate in the human serum (~300 µM). Changes in the renal handling of urate during primate evolution can explain the genetic modification of uricolytic activities in the hominoid lineage without the need of assuming fixation of deleterious mutations.

High-resolution structures of retinol-binding protein in complex with retinol: pH-induced protein structural changes in the crystal state.

The targeted delivery of non-polar ligands by binding proteins to membranes or membrane receptors involves the release of these ligands on or near the plasma membrane of target cells. Because these hydrophobic ligands are often bound inside a deep cavity of binding proteins, as shown previously for plasma retinol-binding protein (RBP), their release from these proteins might require the destabilization of the protein structure by partially denaturing conditions, such as those possibly present near plasma membranes. RBP is a plasma transport protein which delivers specifically retinol from its store sites to target cells. Here, we report the high-resolution (1.1-1.4A) crystal structures of bovine holo-RBP at five different pH values, ranging from 9 to 2. While unraveling details of the native protein structure and of the interactions with retinol at nearly atomic resolution at neutral pH, this study provides evidence for definite pH-induced modifications of several structural features of RBP. The structure most representative of the changes that holo-RBP undergoes at different pH values is that of its flexible state at pH 2. At this pH, most significant are the alteration of the arrangement of salt bridges and of the network of water molecules/H-bonds that participates in the retinol-RBP interaction, an appreciable increase of the volume of the beta-barrel cavity, a considerably higher degree of mobility of the RBP-bound ligand and of several protein regions and the disorder of a large number of solvent molecules that are ordered at neutral pH. These changes are likely to be accompanied by a modification of the pattern of charge distribution on the protein surface. All these changes, which reveal a substantially lowered conformational stability of RBP, presumably occur at the initial stages of the acidic denaturation of RBP and are possibly associated with a facilitated release of the retinol molecule from its carrier protein.