Human low-density lipoprotein receptor plays an important role in hepatitis B virus infection

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV vaccine is effective to prevent new HBV infection but does not offer therapeutic benefit to hepatitis B patients. Neither are current antiviral drugs curative of chronic hepatitis B. A more thorough understanding of HBV infection and replication holds a great promise for identification of novel antiviral drugs and design of optimal strategies towards the ultimate elimination of chronic hepatitis B. Recently, we have developed a robust HBV cell culture system and discovered that human apolipoprotein E (apoE) is enriched on the HBV envelope and promotes HBV infection and production. In the present study, we have determined the role of the low-density lipoprotein receptor (LDLR) in HBV infection. A LDLR-blocking monoclonal antibody potently inhibited HBV infection in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) as well as in primary human hepatocytes. More importantly, small interfering RNAs (siRNAs)-mediated knockdown of LDLR expression and the CRISPR/Cas9-induced knockout of the LDLR gene markedly reduced HBV infection. A recombinant LDLR protein could block heparin-mediated apoE pulldown, suggesting that LDLR may act as an HBV cell attachment receptor via binding to the HBV-associated apoE. Collectively, these findings demonstrate that LDLR plays an important role in HBV infection probably by serving as a virus attachment receptor.     

Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.     

Scrambled isomers as key intermediates in the oxidative folding of ligand binding module 5 of the low density lipoprotein receptor

The ligand binding module five (LA5) of the low density lipoprotein receptor is a small, single-domain protein of 40 residues and three disulfide bonds with a calcium binding motif that is essential for its structure and function. Several mutations in LA5 have been reported to cause familial hypercholesterolemia by impairing a proper folding of the module. The current study reports the oxidative folding and reductive unfolding pathways of wild type and mutant LA5 modules through kinetic and structural analysis of the trapped intermediates. Wild type LA5 folding involves an initial phase of nonspecific packing where the sequential oxidation of its cysteines gives rise to complex equilibrated populations of intermediates. In the presence of calcium, the attainment of a coordination-competent conformation becomes the rate-limiting step of folding while binding of the ion funnels both thermodynamically and kinetically the folding reaction toward the native state. In the absence of calcium, a scrambled isomer (termed Xa) constitutes the global free energy minimum of the folding process. Xa and the native form are stable, inter-convertible species whose relative populations at equilibrium appear displaced in disease-linked mutants toward the scrambled form. Because stable scrambled isomers such as Xa avoid the exposition of reactive cysteines in misfolded modules, they might constitute a strategy to prevent wrong interactions with other domains during folding of the receptor. Comparison of the folding pathways of wild type and mutant LA5 provides the molecular basis to understand how LA modules fold into a functional conformation or upon mutation misfold and lead to disease.     

Phenylalanine 30 plays an important role in receptor binding of verotoxin-1

The homopentameric B subunit of verotoxin 1 (VT1) binds to the glycosphingolipid receptor globotriaosylceramide (Gb₃). We produced mutants with alanine substitutions for residues found near the cleft between adjacent subunits. Substitution of alanine for phenylalanine 30 (Phe-30) resulted in a fourfold reduction in B subunit binding affinity for Gb₃ and a 10-fold reduction in receptor density in a solid-phase binding assay. The interaction of wild-type and mutant B subunits with Pk trisaccharide in solution was examined by titration microcalorimetry. The carbohydrate binding of the mutant was markedly impaired compared with that of the wild type and was too weak to allow calculation of a binding constant. These results demonstrate that the mutation significantly impaired the carbohydrate-binding function of the B subunit. To ensure that the mutation had not caused a significant change in structure, the mutant B subunit was crystallized and its structure was determined by X-ray diffraction. Difference Fourier analysis showed that its structure was identical to that of the wild type, except for the substitution of alanine for Phe-30. The mutation was also produced in the VT1 operon, and mutant holotoxin was purified to homogeneity. The cytotoxicity of the mutant holotoxin was reduced by a factor of 10⁵ compared to that of the wild type in the Vero cell cytotoxicity assay. The results suggest that the aromatic ring of Phe-30 plays a major role in binding of the B subunit to the Galα1-4Galβ1-4Glc trisaccharide portion of Gb₃. Examination of the VT1 B crystal structure suggests two potential carbohydrate-binding sites which lie on either side of Phe-30.     

The structure of the interleukin-15 alpha receptor and its implications for ligand binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.     

Binding of low-density lipoprotein and chylomicron remnants to the hepatic low-density lipoprotein receptor of dogs, rats and rabbits demonstrated by ligand blotting. Failure to detect a distinct chylomicron-remnant-binding protein by ligand blotting

In this paper, human low-density lipoprotein (LDL), rat chylomicron remnants and very-low-density lipoproteins of beta-mobility from cholesterol-fed rabbits (beta VLDL) have been shown to bind strongly to a protein present in solubilised liver membranes of rats, rabbits and dogs by ligand blotting with biotin-modified lipoproteins. This binding protein was identified as the LDL-receptor on several criteria. First, binding of the lipoproteins to the receptor was saturable and Ca2+-dependent; secondly, the apparent relative molecular mass of the binding protein (ranging from 128,000 in the rabbit, 145,000 in the rat to 147,000 in the dog) was similar to that of the purified bovine LDL receptor. Finally, binding activity was greatly increased in the livers of rats treated with oestrogen in pharmacological doses and absent from the liver of Watanabe heritable hyperlipidaemic (WHHL) rabbits that have a genetic defect in the LDL receptor. Some binding was also observed to a high-molecular-mass protein present in solubilised liver membranes of rats and rabbits, which, in rabbits at least, shared antigenic determinants with rabbit apoB and was not likely to be related to the LDL receptor as it was present in equal amounts in normal and WHHL rabbits. No evidence was obtained for a specific chylomicron remnant binding protein, distinct from the LDL receptor, whose activity could be detected in solubilised liver membranes by ligand blotting although a variety of solubilisation and fractionation conditions were employed.     

Immunoblotting and ligand blotting of the low-density lipoprotein receptor of human liver, HepG2 cells and HeLa cells

Low-density lipoprotein receptors from adult human liver and the human hepatoblastoma cell line HepG2 were analyzed by polyacrylamide electrophoresis in SDS followed by immuno- and ligand blotting. In both liver and HepG2 we detected a protein band with apparent relative molecular mass of 130 kDa, which is similar to that of the LDL receptor in fibroblasts. In addition we showed that HeLa cells also possess this LDL-receptor protein.     

The role of dynamics in modulating ligand exchange in intracellular lipid binding proteins

Lipids are essential for many biological processes and crucial in the pathogenesis of several diseases. Intracellular lipid-binding proteins (iLBPs) provide mobile hydrophobic binding sites that allow hydrophobic or amphipathic lipid molecules to penetrate into and across aqueous layers. Thus iLBPs mediate the lipid transport within the cell and participate to a spectrum of tissue-specific pathways involved in lipid homeostasis. Structural studies have shown that iLBPs' binding sites are inaccessible from the bulk, implying that substrate binding should involve a conformational change able to produce a ligand entry portal. Many studies have been reported in the last two decades on iLBPs indicating that their dynamics play a pivotal role in regulating ligand binding and targeted release. The ensemble of reported data has not been reviewed until today. This review is thus intended to summarize and possibly generalize the results up to now described, providing a picture which could help to identify the missing notions necessary to improve our understanding of the role of dynamics in iLBPs' molecular recognition. Such notions would clarify the chemistry of lipid binding to iLBPs and set the basis for the development of new drugs.     

The first epidermal growth factor-like domain of the low-density lipoprotein receptor contains a noncanonical calcium binding site

Removal of cholesterol-containing particles from the circulation is mediated by the low-density lipoprotein (LDL) receptor. Upon ligand binding, the receptor-ligand complex is endocytosed, and the ligand is released. The important biological role of the LDL receptor (LDLR) has been highlighted by the identification of more than 400 LDLR mutations that are associated with familial hypercholesterolemia. The extracellular region of the LDLR is modular in nature and principally comprises multiple copies of ligand binding, epidermal growth factor-like (EGF), and YWTD-type domains. This report describes characterization of the calcium binding properties of the tandem pair of EGF domains. While only the C-terminal EGF module contains the consensus sequence associated with calcium binding, a noncanonical calcium binding site in the N-terminal domain has been revealed using solution NMR spectroscopy. The calcium dissociation constants for the N- and C-terminal sites have been measured under physiologically relevant pH and ionic strength conditions using a combination of solution NMR, intrinsic protein fluorescence, and chromophoric chelator methods to be approximately 50 microM and approximately 10-20 microM, respectively. Identification of the novel calcium binding motif in LDLR sequences from other species suggests that it may confer specificity within the LDLR gene family. Comparison of the K(d) for the C-terminal site with the calcium concentration in late vesicles indicates that the binding properties of this module may be tuned to titrate upon endocytosis of the LDL receptor-ligand complex, and thus calcium binding may play a role in the ligand dissociation process.     

Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low-density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoprotein.

4-Hydroxynonenal (HNE) is a major aldehydic propagation product formed during peroxidation of unsaturated fatty acids. The aldehyde was used to modify freshly prepared human low-density lipoprotein (LDL). A polyclonal antiserum was raised in the rabbit and absorbed with freshly prepared LDL. The antiserum did not react with human LDL, but reacted with CuCl2-oxidized LDL and in a dose-dependent manner with LDL, modified with 1, 2 and 3 mM-HNE, in the double-diffusion analysis. LDL treated with 4 mM of hexanal or hepta-2,4-dienal or 4-hydroxyhexenal or malonaldehyde (4 or 20 mM) did not react with the antiserum. However, LDL modified with 4 mM-4-hydroxyoctenal showed a very weak reaction. Lipoprotein (a) and very-low-density lipoprotein were revealed for the first time to undergo oxidative modification initiated by CuCl2. This was evidenced by the generation of lipid hydroperoxides and thiobarbituric acid-reactive substances, as well as by a marked increase in the electrophoretic mobility. After oxidation these two lipoproteins also reacted positively with the antiserum against HNE-modified LDL.     

Solution structure and dynamics of the central CCP module pair of a poxvirus complement control protein

The complement control protein (CCP) module (also known as SCR, CCP or sushi domain) is prevalent amongst proteins that regulate complement activation. Functional and mutagenesis studies have shown that in most cases two or more neighbouring CCP modules form specific binding sites for other molecules. Hence the orientation in space of a CCP module with respect to its neighbours and the flexibility of the intermodular junction are likely to be critical for function. Vaccinia virus complement control protein (VCP) is a complement regulatory protein composed of four tandemly arranged CCP modules. The solution structure of the carboxy-terminal half of this protein (CCP modules 3 and 4) has been solved previously. The structure of the central portion (modules 2 and 3, VCP approximately 2,3) has now also been solved using NMR spectroscopy at 37 degrees C. In addition, the backbone dynamics of VCP approximately 2,3 have been characterised by analysis of its (15)N relaxation parameters. Module 2 has a typical CCP module structure while module 3 in the context of VCP approximately 2,3 has some modest but significant differences in structure and dynamics to module 3 within the 3,4 pair. Modules 2 and 3 do not share an extensive interface, unlike modules 3 and 4. Only two possible NOEs were identified between the bodies of the modules, but a total of 40 NOEs between the short intermodular linker of VCP approximately 2,3 and the bodies of the two modules determines a preferred, elongated, orientation of the two modules in the calculated structures. The anisotropy of rotational diffusion has been characterised from (15)N relaxation data, and this indicates that the time-averaged structure is more compact than suggested by (1)H-(1)H NOEs. The data are consistent with the presence of many intermodular orientations, some of which are kinked, undergoing interconversion on a 10(-8)-10(-6) second time-scale. A reconstructed representation of modules 2-4 allows visualisation of the spatial arrangement of the 11 substitutions that occur in the more potent complement inhibitor from Variola (small pox) virus.     

The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism

The Anopheles gambiae mosquito is the main vector of malaria transmission in sub-Saharan Africa. We present here a 1.5A crystal structure of AgamOBP1, an odorant binding protein (OBP) from the A. gambiae mosquito. The protein crystallized as a dimer with a unique binding pocket consisting of a continuous tunnel running through both subunits of the dimer and occupied by a PEG molecule. We demonstrate that AgamOBP1 undergoes a pH dependent conformational change that is associated with reduced ligand binding. A predominance of acid-labile hydrogen bonds involving the C-terminal loop suggests a mechanism in which a drop in pH causes C-terminal loop to open, leaving the binding tunnel solvent exposed, thereby lowering binding affinity for ligand. Because proteins from two distantly related insects also undergo a pH dependent conformational change involving the C-terminus that is associated with reduced ligand affinity, our results suggest a common mechanism for OBP activity.     

The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition.

The receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) was purified recently, and its binding of ligand was shown to depend on calcium ions (Moestrup, S. K., and Gliemann, J. (1989) J. Biol. Chem. 264, 15574-15577). This paper shows that the 440-kDa human placental alpha 2MR is a cysteine-rich glycoprotein with high affinity calcium binding sites important for receptor conformation; and the relationship between Ca2+ concentration and receptor function is presented. Autoradiography showed 45Ca2+ binding to the 440-kDa alpha 2MR blotted onto nitrocellulose from a sodium dodecyl sulfate-polyacrylamide gel. alpha 2MR immobilized on nitrocellulose in the absence of sodium dodecyl sulfate bound 45Ca2+ in the presence of 5 mM Mg2+, and 2-3 microM unlabeled Ca2+ was required to displace half of the bound 45Ca2+. The calcium concentration dependence showed upward concave Scatchard plots, and the number of binding sites was estimated to be approximately eight/alpha 2MR molecule. Binding of calcium did not change in the pH range 6.5-8.0 but decreased at lower pH values. Addition of Ca2+ to the medium was necessary for receptor binding of the alpha 2-macroglobulin-trypsin complex, and half of the maximal binding capacity was obtained with about 16 micrograms Ca2+ at pH 7.8. The requirement for calcium was increased at lower pH values, and half of the maximal 125I-alpha 2M-trypsin binding was obtained with about 30-40 microM Ca2+ at pH 7.0. Monoclonal antibodies were produced against alpha 2MR, and one of them distinguished between the Ca2(+)-occupied and nonoccupied forms. Like Ca2+, Sr2+ and Ba2+ elicited ligand binding affinity and competed for binding with 45Ca2+ in the order Ca2+ greater than Sr2+ greater than Ba2+. In conclusion, calcium ions bind specifically to alpha 2MR with high affinity, and it is likely that several sites on the alpha 2MR molecule have to be occupied to elicit the conformation recognizing the ligand.     

Effect of ligand binding and DNA binding on the structure of the mouse oestrogen receptor

We have investigated the effects of ligand and DNA binding on the structure of the oestrogen receptor by performing limited proteolysis and analysing DNA binding activity by gel shift analysis. The effects of oestradiol, 4-hydroxytamoxifen and ICI 164,384 have been examined and we have found that despite differences in the DNA binding activity or relative mobility of the receptor-DNA complex we were unable to detect differences in the cleavage pattern produced by trypsin, chymotrypsin, Staphylococcus aureus V8, papain or elastase. Inhibition of DNA binding by ICI 164,384 was lost in receptor fragments that lacked the hormone binding domain. In contrast to the full-length receptor, proteolytic fragments produced by chymotrypsin differed in their ability to bind to an oestrogen response element (ERE) vs a thyroid response element (TRE). Evidence is presented that this difference can be accounted for by the inability of fragments lacking the hormone binding domain to dimerise on a TRE.     

The role of the endothelium in myocardial lipoprotein dynamics

This overview is presented, in the main, to summarize the following areas of myocardial lipoprotein metabolism: 1. The nature and extent of the cardiac endothelium. 2. The interactions between the endothelium and chylomicrons, very low, low and high density lipoproteins in the presence and absence of lipoprotein lipase. 3. The importance of the endothelial lipoprotein lipase and the mechanisms involved in the enzymes' sequestration at that site. 4. The physiological role of lipoprotein lipase in the provision of oxidizable fuel for the heart.     

Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.