ZG16p, an animal homolog of β-prism fold plant lectins, interacts with heparan sulfate proteoglycans in pancreatic zymogen granules

ZG16p is a soluble 16?kDa pancreatic protein having structural similarities with plant β-prism fold lectins such as the banana lectin BanLec and the jackfruit lectin jacalin. ZG16p is postulated to be involved in the formation of zymogen granules by interacting with proteoglycans (PGs) localized in pancreatic exocrine granule membranes, but direct evidence was lacking. We characterized the structural properties of rat pancreatic zymogen granule PGs and examined their interaction with ZG16p. Structural analysis of the glycosaminoglycans (GAGs) showed that rat pancreatic zymogen granule PGs have heparan sulfate chains with a unique property, a high degree of sulfation (ΔUA-GlcNAc:ΔUA-GlcNS:ΔUA-GlcNAc6S:ΔUA-GlcNS6S:ΔUA2S-GlcNS:ΔUA2S-GlcNS6S, 27.9:16.6:5.7:22.5:6.2:21.1). After heparin lyase II digestion, the core proteins derived from the PGs were detected at molecular weights of 66,000 and 35,000-40,000. An overlay binding assay revealed that ZG16p binds specifically to heparan sulfate PGs by recognizing their GAG chains. Affinity chromatography demonstrated that ZG16p binds most strongly to heparin among the zymogen granule proteins. Site-directed mutational analysis revealed that the basic amino acid residues located in two putative carbohydrate-binding sites (CBSs) of ZG16p, which were found in association with the crystal structure of BanLec, are responsible for the recognition of heparin. These observations suggest that ZG16p is the primary binding partner of the granule heparan sulfate PGs. ZG16p may cross-link the granule heparan sulfate chains via two CBSs and facilitate the formation of a submembranous matrix, a sorting platform for enzyme proteins on the luminal side of the zymogen granule membrane.     

Crystal structure of a β-prism II lectin from Remusatia vivipara

The crystal structure of a β-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4??. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.     

Crystal structures of human Ero1�� reveal the mechanisms of regulated and targeted oxidation of PDI

In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine‐tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b′‐domain through its substrate‐binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.     

Crystal structures of Trichoderma reesei β-galactosidase reveal conformational changes in the active site

We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal) at a 1.2? resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4? resolutions, respectively. Tr-β-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-β-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-β-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain.     

Crystal structures of a stabilized β1-adrenoceptor bound to the biased agonists bucindolol and carvedilol

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Highlights? Structures of β1AR bound to the biased agonists bucindololol and carvedilol ? The biased agonists form unique contacts with β1AR not seen with other antagonists ? The structures explain the pharmacological differences in the Arg389Gly polymorphism     

Crystal structures of rare disaccharides, α-D-glucopyranosyl β-D-psicofuranoside, and α-D-galactopyranosyl β-D-psicofuranoside

The crystal structures of α-D-glucopyranosyl β-D-psicofuranoside and α-D-galactopyranosyl β-D-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R(1)=0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between (4)E and (4)T(3). Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Crystal and molecular structures of substituted α-d-glucopyranosides

The crystal and molecular structures of methyl 2,4,6-tri-O-pivaloyl-α-d-glucopyranoside (1), methyl 4,6-O-(R)-benzylidene-2-O-pivaloyl-α-d-glucopyranoside (2), and methyl 4,6-O-(R)-benzylidene-2,3-di-O-pivaloyl-α-d-glucopyranoside (3) were determined by X-ray analysis. Crystals of 1 are orthorhombic, space group P2₁2₁2₁ with the unit cell a = 13.026(2), b = 16.832, c = 11.929(2) Å, Z = 4. Crystals of 2 are monoclinic, space group P2₁. The unit-cell parameters are a = 6.519(1), b = 14.664(4), c = 10.635(4) Å, β = 93.18(1)°, Z = 2. Crystals of 3 are orthorhombic, space group P2₁2₁2₁ with a = 10.006(3), b = 13.874(3), c = 18.527(5) Å, Z = 4. The structures were solved by MULTAN and refined by a full-matrix procedure to final values of R = 0.084 (1), 0.048 (2), and 0.069 (3). The pyranose ring in each compound adopts the ⁴C₁ conformation. The 1,3-dioxane rings in 2 and 3 show a chair conformation. The molecular packing in 1 is through the hydrogen bonds involving HO-3 and the 6-O-pivaloyl carbonyl group [HO-3 ⋯ O-9, 2.855(8) Å], which connect the molecules into a chain along

. The endocyclic oxygen atom is involved in an intermolecular hydrogen-bond with HO-3 [2.848(4) Å], joining molecules of 2 into the chains along

. There are no free hydroxyl groups in 3 and molecular packing reflects van der Waals interactions only.     

Two immunoglobulin tandem proteins with a linking β-strand reveal unexpected differences in cooperativity and folding pathways

The study of the folding of single domains, in the context of their multidomain environment, is important because more than 70% of eukaryotic proteins are composed of multiple domains. The structures of the tandem immunoglobulin (Ig) domain pairs A164-A165 and A168-A169, from the A-band of the giant muscle protein titin, reveal that they form tightly associated domain arrangements, connected by a continuous β-strand. We investigate the thermodynamic and kinetic properties of these tandem domain pairs. While A164-A165 apparently behaves as a single cooperative unit at equilibrium, unfolding without the accumulation of a large population of intermediates, domains in A168-A169 behave independently. Although A169 appears to be stabilized in the tandem protein, we show that this is due to nonspecific stabilization by extension. We elucidate the folding and unfolding pathways of both tandem pairs and show that cooperativity in A164-A165 is a manifestation of the relative refolding and unfolding rate constants of each individual domain. We infer that the differences between the two tandem pairs result from a different pattern of interactions at the domain/domain interface.     

The β2 subunit of human placenta hexosaminidase (Hex) A and B is a non-random association of the two polypeptides αa and βb

The subunit structures of placental Hex A and B have previously been assigned as _a and _b, respectively. The ₂ subunit is composed of two non-identical polypeptide chains, _a and _b. Purified Hex A and B were fractionated on a chromatofocusing column, and the fractions were reduced and then alkylated with iodo-I-¹⁴C-acetamide. The polypeptide chains were separated by polyacrylamide-gel isoelectric focusing. From the radioactivity measurements of the polypeptides a constant value for ₂ was obtained in all the chromatofocusing fractions, demonstrating a non-random structure of (₂) in each ₂ subunit.     

Characterization of a panel of mouse single-chain antibodies against human recombinant interferon β1b

A panel of single-chain Fv-antibodies (ScFv’s) against recombinant human interferon beta 1b (rhIFN-β1b) has been obtained from immune and naïve combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv’s were expressed in Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv’s in periplasm and incubation medium as well as their expression and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv’s sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of theirs variable domains has been shown. For the ScFv’s isolated from the immune library, specificity of their binding with native and denatured rhIFN-β1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv’s panel were in the range from 1.96 × 10^?8 to 1.69 × 10^?9 M.     

Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose

We solved the 1.8 ? crystal structure of β-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of β-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed β-propeller and a C-terminal β-sandwich module. The active site is located in the centre of the β-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 β-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 ? crystal structure of the β-fructofuranosidase complex with β-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.     

Quaternary association in β-prism I fold plant lectins: Insights from X-ray crystallography, modelling and molecular dynamics

Dimeric banana lectin and calsepa, tetrameric artocarpin and octameric heltuba are mannose-specific beta-prism I fold lectins of nearly the same tertiary structure. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions that undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Energy minimization and simulations were also carried out on models using all possible oligomers by employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also led to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding.     

Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP

The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 ?. The enzyme is a homodimer with a molecular mass of ～ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof?' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: ? -transaminases and -transaminases bind by dynamic light scattering (View interaction) ? -transaminase and -transaminase bind by x-ray crystallography (View interaction) ? -transaminase and -transaminase bind by x-ray crystallography (View interaction).     

Expression and purification of coronavirus envelope proteins using a modified β-barrel construct

Coronavirus envelope (E) proteins are short (～100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2-3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides.     

Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization

A β-glucosidase gene (bglI) from Trichoderma reesei was cloned into the pPIC9 vector and integrated into the genome of Pichia pastoris GS115. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter and using Saccharomyces cerevisiae secretory signal peptide (α-factor), the recombinant β-glucosidase was expressed and secreted into the culture medium. The maximum recombinant β-glucosidase activity achieved was 60 U/ml, and β-glucosidase expression reached 0.3 mg/ml. The recombinant 76 kDa β-glucosidase was purified 1.8-fold with 26% yield and a specific activity of 197 U/mg. It was optimally active at 70°C and pH 5.0.     

Structural genomics of human proteins – target selection and generation of a public catalogue of expression clones

Background  

The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia colihost cells.     

Crystal structure of human β-galactosidase: structural basis of Gm1 gangliosidosis and morquio B diseases

G(M1) gangliosidosis and Morquio B are autosomal recessive lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme β-d-galactosidase (β-Gal), which lead to accumulations of the β-Gal substrates, G(M1) ganglioside, and keratan sulfate. β-Gal is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. This study shows the crystal structures of human β-Gal in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. Human β-Gal is composed of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. To gain structural insight into the molecular defects of β-Gal in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.     

Biochemical Properties and Crystal Structure of a β-Phenylalanine Aminotransferase from Variovorax paradoxus

By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg⁻¹ for (S)-β-phenylalanine at 30°C and 33 U mg⁻¹ at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.     

Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin-Dendra2 fusion proteins

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin-Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.