Structural characterization of a Vatairea macrocarpa lectin in complex with a tumor-associated antigen: A new tool for cancer research.

Legume lectins are the most thoroughly studied group of lectins and have been widely linked to many pathological processes. Their use as immunohistochemistry markers for cell profiling and cancer diagnosis have made these molecules important tools for immunological studies and have stimulated the prospection and characterization of new lectins. The crystal structures of a recombinant seed lectin from Vatairea macrocarpa (rVML) and its complexes with GalNAcα1-O-Ser, GalNAc and α-lactose, have been determined at 1.90, 1.97, 2.70 and 1.83Å resolution, respectively. Small angle X-ray scattering and calorimetry assays have confirmed the same pH stable oligomerization pattern and binding profiles proposed for its wild-type counterpart. In silico analyzes have explored the potential of this recombinant lectin as new tool for cancer research through a comparative profile with other legume lectins widely used for cancer diagnosis and prognosis. The results suggest the recognition of specific epitopes exhibited on different cancer cells as a process that relies on the disposition of hydrophobic clusters and charged regions around the lectin carbohydrate-binding site, favouring the anchorage of different groups in the antigen boundaries, highlighting the different potential of each analyzed lectin. In conclusion, the experimental results and comparative analysis show that rVML is as a promising tool for cancer research, able to bind with high affinity specific tumor-associated antigens, highly stable and easily produced.

High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins.

Plant lectins have been studied as histological markers and promising antineoplastic molecules for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been determined at the resolution of 1.4Å and 1.7Å, respectively. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.

Structural studies of an anti-inflammatory lectin from Canavalia boliviana seeds in complex with dimannosides.

Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.

Crystal structure of Dioclea violacea lectin and a comparative study of vasorelaxant properties with Dioclea rostrata lectin.

Lectins from Diocleinae subtribe belong to the family of legume lectins and are characterized by high identity between their amino acids sequences. It has been shown that punctual differences in amino acid sequences, such as one single amino acid or an alternative conformation, represent changes in biological activities caused by these lectins. Therefore, a more detailed understanding of three-dimensional structures of these proteins is essential for accurate analyzing the relationship between structure and function. In this study lectins purified from the seeds of Dioclea violacea (DVL) and Dioclea rostrata (DRL) were compared with regard to crystal structure and vasorelaxant properties. Differences in structure of lectins were found to be reflected in differences in vasorelaxant effects based on their high specificity and selectivity for cell glycans. Binding activity was related to the position of specific residues in the carbohydrate recognition domain (CRD). DVL complexed structure was solved by X-ray crystallography and was compared to native DVL and DRL. Therefore, DVL was co-crystallized with X-Man, and a molecular modeling with X-Man complexed with DVL was done to compare the complexed and native forms adjusted fit. The relatively narrow and deep CRD in DVL promotes little interaction with carbohydrates; in contrast, the wider and shallower CRD in DRL favors interaction. This seems to explain differences in the level of relaxation induced by DVL (43%) and DRL (96%) in rat aortic rings.

Crystallization and preliminary X-ray diffraction of the surfactant protein Lv-ranaspumin from the frog Leptodactylus vastus.

Lv-ranaspumin is a natural surfactant protein with a molecular mass of 23.5 kDa which was isolated from the foam nest of the frog Leptodactylus vastus. Only a partial amino-acid sequence is available for this protein and it shows it to be distinct from any protein sequence reported to date. The protein was purified from the natural source by ion-exchange and size-exclusion chromatography and was crystallized by sitting-drop vapour diffusion using the PEG/Ion screen at 293 K. A complete data set was collected to 3.5 Å resolution. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.96, b = 89.99, c = 106.00 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54%.

Crystal structure of a pro-inflammatory lectin from the seeds of Dioclea wilsonii Standl.

The crystal structure and pro-inflammatory property of a lectin from the seeds of Dioclea wilsonii (DwL) were analyzed to gain a better understanding of structure/function relationships of Diocleinae lectins. Following crystallization and structural determination by standard molecular replacement techniques, DwL was found to be a tetramer based on PISA analysis, and composed by two metal-binding sites per monomer and loops which are involved in molecular oligomerization. DwL presents 96% and 99% identity with two other previously described lectins of Dioclea rostrata (DRL) and Dioclea grandiflora (DGL). DwL differs structurally from DVL and DRL with regard to the conformation of the carbohydrate recognition domain and related biological activities. The structural analysis of DwL in comparison to other Diocleinae lectins can be related to the differences in the dose-dependent pro-inflammatory effect elicited in Wistar rats, probably via specific interactions with mast cells complex carbohydrate, resulting in significant paw edema. DwL appears to be involved in positive modulation of mast cell degranulation via recognition of surface carbohydrates. Since this recognition is dependent on site volume and CRD configuration, edematogenesis mediated by resident cells varies in potency and efficacy among different Diocleinae lectins.

Structural determinants of the enantioselectivity of the hydroxynitrile lyase from Hevea brasiliensis.

The hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis (HbHNL) is utilized as a biocatalyst in stereospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones. The catalyzed reaction represents one of the few industrially relevant examples of enzyme mediated C-C coupling reactions. In this work, we determined the X-ray crystal structures (at 1.54 and 1.76 Angstroms resolution) of HbHNL complexes with two chiral substrates -- mandelonitrile and 2,3-dimethyl-2-hydroxy-butyronitrile -- by soaking and rapid freeze quenching techniques. This is the first structural observation of the complex between a HNL and chiral substrates. Consistent with the known selectivity of the enzyme, only the S-enantiomers of the two substrates were observed in the active site. The binding modes of the chiral substrates were identical to that observed for the biological substrate acetone cyanohydrin. This indicates that the transformation of these non-natural substrates follows the same mechanism. A large hydrophobic pocket was identified in the active site of HbHNL which accommodates the more voluminous substituents of the two substrates. A three-point binding mode of the substrates -- hydrophobic pocket, hydrogen bonds between the hydroxyl group and Ser80 and Thr11, electrostatic interaction of the cyano group with Lys236 -- offers a likely structural explanation for the enantioselectivity of the enzyme. The structural data rationalize the observed (S)-enantioselectivity and form the basis for modifying the stereospecificity through rational design. The structures also revealed the necessity of considerable flexibility of the sidechain of Trp128 in order to bind and transform larger substrates.

Long-distance gene flow and cross-Andean dispersal of lowland rainforest bees (Apidae: Euglossini) revealed by comparative mitochondrial DNA phylogeography.

Euglossine bees (Apidae; Euglossini) exclusively pollinate hundreds of orchid species and comprise up to 25% of bee species richness in neotropical rainforests. As one of the first studies of comparative phylogeography in a neotropical insect group, we performed a mitochondrial DNA (mtDNA)-based analysis of 14 euglossine species represented by populations sampled across the Andes and/or across the Amazon basin. The mtDNA divergences within species were consistently low; across the 12 monophyletic species the mean intraspecific divergence among haplotypes was 0.9% (range of means, 0-1.9%). The cytochrome oxidase 1 (CO1) divergence among populations separated by the Andes (N = 11 species) averaged 1.1% (range 0.0-2.0%). The mtDNA CO1 data set displayed homogeneous rates of nucleotide substitution, permitting us to infer dispersal across the cordillera long after the final Andean uplift based on arthropod molecular clocks of 1.2-1.5% divergence per million years. Gene flow across the 3000-km breadth of the Amazon basin was inferred from identical cross-Amazon haplotypes found in five species. Although mtDNA haplotypes for 12 of the 14 euglossine species were monophyletic, a reticulate CO1 phylogeny was recovered in Euglossa cognata and E. mixta, suggesting large ancestral populations and recent speciation. Reference to closely related outgroups suggested recent speciation for the majority of species. Phylogeographical structure across a broad spatial scale is weaker in euglossine bees than in any neotropical group previously examined, and may derive from a combination of Quaternary speciation, population expansion and/or long-distance gene flow.

Observation of a short, strong hydrogen bond in the active site of hydroxynitrile lyase from Hevea brasiliensis explains a large pKa shift of the catalytic base induced by the reaction intermediate.

The hydroxynitrile lyase from Hevea brasiliensis (HbHNL) uses a catalytic triad consisting of Ser(80)-His(235)-Asp(207) to enhance the basicity of Ser(80)-O gamma for abstracting a proton from the OH group of the substrate cyanohydrin. Following the observation of a relatively short distance between a carboxyl oxygen of Asp(207) and the N delta(1)(His(235)) in a 1.1 A crystal structure of HbHNL, we here show by (1)H and (15)N-NMR spectroscopy that a short, strong hydrogen bond (SSHB) is formed between the two residues upon binding of the competitive inhibitor thiocyanate to HbHNL: the proton resonance of H-N delta 1(His(235)) moves from 15.41 ppm in the free enzyme to 19.35 ppm in the complex, the largest downfield shift observed so far upon inhibitor binding. Simultaneously, the D/H fractionation factor decreases from 0.98 to 0.35. In the observable pH range, i.e. between pH 4 and 10, no significant changes in chemical shifts (and therefore hydrogen bond strength) were observed for free HbHNL. For the complex with thiocyanate, the 19.35 ppm signal returned to 15.41 ppm at approximately pH 8, which indicates a pK(a) near this value for the H-N epsilon(2)(His(235)). These NMR results were analyzed on the basis of finite difference Poisson-Boltzmann calculations, which yielded the relative free energies of four protonation states of the His(235)-Asp(207) pair in solution as well as in the protein environment with and without bound inhibitor. The calculations explain all the NMR features, i.e. they suggest why a short, strong hydrogen bond is formed upon inhibitor binding and why this short, strong hydrogen bond reverts back to a normal one at approximately pH 8. Importantly, the computations also yield a shift of the free energy of the anionic state relative to the zwitterionic reference state by about 10.6 kcal/mol, equivalent to a shift in the apparent pK(a) of His(235) from 2.5 to 10. This huge inhibitor-induced increase in basicity is a prerequisite for His(235) to act as general base in the HbHNL-catalyzed cyanohydrin reaction.