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.

Structure and properties of the oyster mushroom (Pleurotus ostreatus) lectin.

Pleurotus ostreatus Lectin (POL) is a 353 amino acid chain lectin that can be purified from the fruiting bodies of the very well-known and widely diffused edible oyster mushrooms (P. ostreatus). The lectin has been partially characterized by different groups and, although it was crystallized about 20 years ago, its 3D structure and the details of its interactions with carbohydrates are still unknown. This paper reports the 3D structure and ligand-binding properties of POL. We have determined the X-ray structure of the apo-protein purified from the fruiting bodies of the mushroom and that of the recombinant protein in complex with melibiose to a resolution of about 2 Å. The lectin is a homodimer in which the two polypeptide chains are linked by a disulfide bridge. A POL monomer is composed of two highly homologous ß-jellyroll domains each of which containing a calcium-dependent carbohydrate-binding site. A high degree of sequence similarity is observed between the two carbohydrate-binding modules present in each monomer. The structure of the lectin in complex with melibiose reveals that a POL dimer has four calcium-dependent carbohydrate-binding sites. The interaction with sugars in solution has been characterized by isothermal titration calorimetry and saturation transfer difference NMR and it sheds new light on the molecular determinants of POL specificity. The lectin exhibits in vitro antiproliferative effects against human cancer cell lines and presents structural similarity with the prototype member of the CBM67 family, the noncatalytic domain of Streptomyces avermitilis α-rhamnosidase.

Encapsulation of Photosystem I in Organic Microparticles Increases Its Photochemical Activity and Stability for Ex Vivo Photocatalysis.

Photosystem I (PSI) is a pigment binding multisubunit protein complex involved in the light phase of photosynthesis, catalyzing a light-dependent electron transfer reaction from plastocyanin to ferredoxin. PSI is characterized by a photochemical efficiency close to one, suggesting its possible application in light-dependent redox reaction in an extracellular context. The stability of PSI complexes isolated from plant cells is however limited if not embedded in a protective environment. Here we show an innovative solution for exploiting the photochemical properties of PSI, by encapsulation of isolated PSI complexes in PLGA (poly lactic-co-glycolic acid) organic microparticles. These encapsulated PSI complexes were able to catalyze light-dependent redox reactions with electron acceptors and donors outside the PLGA microparticles. Moreover, PSI complexes encapsulated in PLGA microparticles were characterized by a higher photochemical activity and stability compared with PSI complexes in detergent solution, suggesting their possible application for ex vivo photocatalysis.

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.