Structure and properties of the giant reed (Arundo donax) lectin (ADL).

Arundo donax lectin (ADL) is a 170 amino acid protein that can be purified from the rhizomes of the giant reed or giant cane by exploiting its selective binding to chitin followed by elution with N-acetylglucosamine. The lectin is listed in the UniProt server, the largest protein sequence database, as an uncharacterized protein with chitin-binding domains (A0A0A9P802). This paper reports the purification, structure and ligand-binding properties of ADL. The lectin is a homodimer in which the two protomers are linked by two disulfide bridges. Each polypeptide chain presents four carbohydrate-binding modules that belong to carbohydrate-binding module family 18. A high degree of sequence similarity is observed among the modules present in each protomer. We have determined the X-ray structure of the apo-protein to a resolution of 1.70 Å. The carbohydrate-binding modules, that span a sequence of approximately 40 amino acids, present four internal disulfide bridges, a very short antiparallel central beta sheet and three short alpha helices, two on one side of the beta sheet and one on the other. The structures of the complexes of the lectin with N-acetylglucosamine, N-acetyllactosamine, N-acetylneuraminic acid and N-N'diacetylchitobiose reveal that ADL has two primary and two secondary carbohydrate-binding sites per dimer. They are located at the interface between the two protomers, and each binding site involves residues of both chains. The lectin presents structural similarity to the wheat germ agglutinin family, in particular, to isoform 3.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1.

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel ß-sheets of seven and six strands, arranged as a distorted ß-sandwich. Furthermore, two long loops partially cover the concave face of the ß-sandwich defined by the ß-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Extrinsic Functions of Lectin Domains in O-N-Acetylgalactosamine Glycan Biosynthesis.

Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (ß-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate. T3lec, but not T2lec and T4lec, had a clear activating effect on Drosophila melanogaster core 1 galactosyltransferase enzyme activity and a predominant inhibitory effect on in vivo human core 1 glycan biosynthesis. The regulatory role of the ß-trefoil fold of ppGalNAc-Ts in enzymatic activity of glycosyltransferases involved in the O-glycan biosynthesis pathway, described here for the first time, helps clarify the mechanism of biosynthesis of complex biopolymers (such as glycans) that is not template-driven.

Enhancement by GOSPEL protein of GAPDH aggregation induced by nitric oxide donor and its inhibition by NAD(.).

Glyceraldehyde-3-phosphate dehydrogenase's (GAPDH's) competitor of Siah Protein Enhances Life (GOSPEL) is the protein that competes with Siah1 for binding to GAPDH under NO-induced stress conditions preventing Siah1-bound GAPDH nuclear translocation and subsequent apoptosis. Under these conditions, GAPDH may also form amyloid-like aggregates proposed to be involved in cell death. Here, we report the in vitro enhancement by GOSPEL of NO-induced GAPDH aggregation resulting in the formation GOSPEL-GAPDH co-aggregates with some amyloid-like properties. Our findings suggest a new function for GOSPEL, contrasting with its helpful role against the apoptotic nuclear translocation of GAPDH. NAD(+) inhibited both GAPDH aggregation and co-aggregation with GOSPEL, a hitherto undescribed effect of the coenzyme against the consequences of oxidative stress.

Three-dimensional structure and ligand-binding site of carp fishelectin (FEL).

Carp FEL (fishelectin or fish-egg lectin) is a 238-amino-acid lectin that can be purified from fish eggs by exploiting its selective binding to Sepharose followed by elution with N-acetylglucosamine. Its amino-acid sequence and other biochemical properties have previously been reported. The glycoprotein has four disulfide bridges and the structure of the oligosaccharides linked to Asn27 has been described. Here, the three-dimensional structures of apo carp FEL (cFEL) and of its complex with N-acetylglucosamine determined by X-ray crystallography at resolutions of 1.35 and 1.70 Å, respectively, are reported. The molecule folds as a six-bladed ß-propeller and internal short consensus amino-acid sequences have been identified in all of the blades. A calcium atom binds at the bottom of the funnel-shaped tunnel located in the centre of the propeller. Two ligand-binding sites, α and ß, are present in each of the two protomers in the dimer. The first site, α, is closer to the N-terminus of the chain and is located in the crevice between the second and the third blades, while the second site, ß, is located between the fourth and the fifth blades. The amino acids that participate in the contacts have been identified, as well as the conserved water molecules in all of the sites. Both sites can bind the two anomers, α and ß, of N-acetylglucosamine, as is clearly recognizable in the electron-density maps. The lectin presents sequence homology to members of the tachylectin family, which are known to have a function in the innate immune system of arthropods, and homologous genes are present in the genomes of other fish and amphibians. This structure is the first of a protein of this group and, given the degree of homology with other members of the family, it is expected that it will be useful to experimentally determine other crystal structures using the coordinates of cFEL as a search probe in molecular replacement.

High-resolution structures of mutants of residues that affect access to the ligand-binding cavity of human lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerization of the 9,11-endoperoxide group of PGH2 (prostaglandin H2) to produce PGD2 (prostaglandin D2) with 9-hydroxy and 11-keto groups. The product of the reaction, PGD2, is the precursor of several metabolites involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as ß-trace protein, the second most abundant protein in human cerebrospinal fluid. Previous structural work on the mouse and human proteins has focused on the identification of the amino acids responsible and the proposal of a mechanism for catalysis. In this paper, the X-ray structures of the apo and holo forms (bound to PEG) of the C65A mutant of human L-PGDS at 1.40 Šresolution and of the double mutant C65A/K59A at 1.60 Šresolution are reported. The apo forms of the double mutants C65A/W54F and C65A/W112F and the triple mutant C65A/W54F/W112F have also been studied. Mutation of the lysine residue does not seem to affect the binding of PEG to the ligand-binding cavity, and mutation of a single or both tryptophans appears to have the same effect on the position of these two aromatic residues at the entrance to the cavity. A solvent molecule has also been identified in an invariant position in the cavity of virtually all of the molecules present in the nine asymmetric units of the crystals that have been examined. Taken together, these observations indicate that the residues that have been mutated indeed appear to play a role in the entrance-exit process of the substrate and/or other ligands into/out of the binding cavity of the lipocalin.

BEL ß-trefoil: a novel lectin with antineoplastic properties in king bolete (Boletus edulis) mushrooms.

A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as ß-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) ß-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galß1-3GalNAc, was examined in detail. All the three potential binding sites present in the ß-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.

Structural and biochemical insight into glycogenin inactivation by the glycogenosis-causing T82M mutation.

The X-ray structure of rabbit glycogenin containing the T82M (T83M according to previous authors amino acid numbering) mutation causing glycogenosis showed the loss of Thr82 hydrogen bond to Asp162, the residue involved in the activation step of the glucose transfer reaction mechanism. Autoglucosylation, maltoside transglucosylation and UDP-glucose hydrolyzing activities were abolished even though affinity and interactions with UDP-glucose and positioning of Tyr194 acceptor were conserved. Substitution of Thr82 for serine but not for valine restored the maximum extent of autoglucosylation as well as transglucosylation and UDP-glucose hydrolysis rate. Results provided evidence sustaining the essential role of the lost single hydrogen bond for UDP-glucose activation leading to glycogenin-bound glycogen primer synthesis.

Mechanisms of monomeric and dimeric glycogenin autoglucosylation.

Initiation of glucose polymerization by glycogenin autoglucosylation at Tyr-194 is required to prime de novo biosynthesis of glycogen. It has been proposed that the synthesis of the primer proceeds by intersubunit glucosylation of dimeric glycogenin, even though it has not been demonstrated that this mechanism is responsible for the described polymerization extent of 12 glucoses produced by the dimer. We reported previously the intramonomer glucosylation capability of glycogenin without determining the extent of autoglucopolymerization. Here, we show that the maximum specific autoglucosylation extent (MSAE) produced by the non-glucosylated glycogenin monomer is 13.3 ± 1.9 glucose units, similar to the 12.5 ± 1.4 glucose units measured for the dimer. The mechanism and capacity of the dimeric enzyme to carry out full glucopolymerization were also evaluated by construction of heterodimers able to glucosylate exclusively by intrasubunit or intersubunit reaction mechanisms. The MSAE of non-glucosylated glycogenin produced by dimer intrasubunit glucosylation was 16% of that produced by the monomer. However, partially glucosylated glycogenin was able to almost complete its autoglucosylation by the dimer intrasubunit mechanism. The MSAE produced by heterodimer intersubunit glucosylation was 60% of that produced by the wild-type dimer. We conclude that both intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation. The full glucopolymerization capacity of monomeric glycogenin indicates that the enzyme is able to synthesize the glycogen primer without the need for prior dimerization.

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galß1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.

Evidence for glycogenin autoglucosylation cessation by inaccessibility of the acquired maltosaccharide.

Glycogenin initiates the biosynthesis of proteoglycogen, the mammalian glycogenin-bound glycogen, by intramolecular autoglucosylation. The incubation of glycogenin with UDP-glucose results in formation of a tyrosine-bound maltosaccharide, reaching maximum polymerization degree of 13 glucose units at cessation of the reaction. No exhaustion of the substrate donor occurred at the autoglucosylation end and the full autoglucosylated enzyme continued catalytically active for transglucosylation of the alternative substrate dodecyl-maltose. Even the autoglucosylation cessation once glycogenin acquired a mature maltosaccharide moiety, proteoglycogen and glycogenin species ranging rM 47-200kDa, derived from proteoglycogen, showed to be autoglucosylable. The results describe for the first time the ability of polysaccharide-bound glycogenin for intramolecular autoglucosylation, providing evidence for cessation of the glucose polymerization initiated into the tyrosine residue, by inaccessibility of the acquired maltosaccharide moiety to further autoglucosylation.

The intramolecular autoglucosylation of monomeric glycogenin.

The ability of monomeric glycogenin to autoglucosylate by an intramolecular mechanism of reaction is described using non-glucosylated and partially glucosylated recombinant glycogenin. We determined that monomer glycogenin exists in solution at concentration below 0.60-0.85 microM. The specific autoglucosylation rate of non-glucosylated and glucosylated monomeric glycogenin represented 50 and 70% of the specific rate of the corresponding dimeric glycogenin species. The incorporation of a unique sugar unit into the tyrosine hydroxyl group of non-glucosylated glycogenin, analyzed by autoxylosylation, occurred at a lower rate than the incorporation into the glucose hydroxyl group of the glucosylated enzyme. The intramonomer autoglucosylation mechanism here described for the first time, confers to a just synthesized glycogenin molecule the capacity to produce maltosaccharide primer for glycogen synthase, without the need to reach the concentration required for association into the more efficient autoglucosylating dimer. The monomeric and dimeric interconversion determining the different autoglucosylation rate, might serve as a modulation mechanism for the de novo biosynthesis of glycogen at the initial glucose polymerization step.

Crystal structure of the anticarcinogenic Bowman-Birk inhibitor from snail medic (Medicago scutellata) seeds complexed with bovine trypsin.

The structure of the ternary complex of the anticarcinogenic Bowman-Birk protease inhibitor purified from snail medic (Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0 A. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2' residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29.

Structure and properties of the C-terminal domain of insulin-like growth factor-binding protein-1 isolated from human amniotic fluid.

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.

The antineoplastic lectin of the common edible mushroom (Agaricus bisporus) has two binding sites, each specific for a different configuration at a single epimeric hydroxyl.

The lectin from the common mushroom Agaricus bisporus, the most popular edible species in Western countries, has potent antiproliferative effects on human epithelial cancer cells, without any apparent cytotoxicity. This property confers to it an important therapeutic potential as an antineoplastic agent. The three-dimensional structure of the lectin was determined by x-ray diffraction. The protein is a tetramer with 222 symmetry, and each monomer presents a novel fold with two beta sheets connected by a helix-loop-helix motif. Selectivity was studied by examining the binding of four monosaccharides and seven disaccharides in two different crystal forms. The T-antigen disaccharide, Galbeta1-3GalNAc, mediator of the antiproliferative effects of the protein, binds at a shallow depression on the surface of the molecule. The binding of N-acetylgalactosamine overlaps with that moiety of the T antigen, but surprisingly, N-acetylglucosamine, which differs from N-acetylgalactosamine only in the configuration of epimeric hydroxyl 4, binds at a totally different site on the opposite side of the helix-loop-helix motif. The lectin thus has two distinct binding sites per monomer that recognize the different configuration of a single epimeric hydroxyl. The structure of the protein and its two carbohydrate-binding sites are described in detail in this study.

Crystal structure of chicken liver basic fatty acid-binding protein complexed with cholic acid.

Two paralogous groups of liver fatty acid-binding proteins (FABPs) have been described: the mammalian type liver FABPs and the basic type (Lb-FABPs) characterized in several vertebrates but not in mammals. The two groups have similar sequences and share a highly conserved three-dimensional structure, but their specificity and stoichiometry of binding are different. The crystal structure of chicken Lb-FABP complexed with cholic acid and that of the apoprotein refined to 2.0 A resolution are presented in this paper. The two forms of the protein crystallize in different space groups, and significant changes are observed between the two conformations. The holoprotein binds two molecules of cholate in the interior cavity, and the contacts observed between the two ligands can help to explain the reason for this stoichiometry of binding. Most of the amino acids involved in ligand binding are conserved in other members of the Lb-FABP family. Since the amino acid sequence of the Lb-FABPs is more similar to that of the bile acid-binding proteins than to that of the L-FABPs, the possibility that the Lb-FABPs might be more appropriately called liver bile acid-binding proteins (L-BABPs) is suggested.

Crystallization and preliminary X-ray study of the common edible mushroom (Agaricus bisporus) lectin.

The lectin from the common edible mushroom Agaricus bisporus (ABL) belongs to the group of proteins that have the property of binding the Thomsen-Friedenreich antigen (T-antigen) selectively and with high affinity, but does not show any sequence similarity to the other proteins that share this property. The ABL sequence is instead similar to those of members of the saline-soluble fungal lectins, a protein family with pesticidal properties. The presence of different isoforms has been reported. It has been found that in order to be able to grow diffraction-quality crystals of the lectin, it is essential to separate the isoforms, which was performed by preparative isoelectric focusing. Using standard procedures, it was possible to crystallize the most basic of the forms by either vapour diffusion or equilibrium dialysis, but attempts to grow crystals of the other more acidic forms were unsuccessful. The ABL crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 93.06, b = 98.16, c = 76.38 A, and diffract to a resolution of 2.2 A on a conventional source at room temperature. It is expected that the solution of this structure will yield further valuable information on the differences in the T-antigen-binding folds and will perhaps help to clarify the details of the ligand binding to the protein.