Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3'-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.

• Keywords
• lectins, molecular dynamics, non-canonical amino acids, protein engineering, synthetic glycobiology,
• MeSH
• Galectin 1 * chemistry   MeSH
• Galectins metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Oligosaccharides chemistry   MeSH
• Tryptophan * MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Galectin 1 * MeSH
• Galectins MeSH
• Ligands MeSH
• Oligosaccharides MeSH
• Tryptophan * MeSH

Carbohydrate-binding proteins from pathogenic bacteria and fungi have been shown to be implicated in various pathological processes, where they interact with glycans present on the surface of the host cells. These interactions are part of the initial processes of infection of the host and are very important to study at the atomic level. Here, we report the room temperature neutron structures of PLL lectin from Photorhabdus laumondii in its apo form and in complex with deuterated L-fucose, which is, to our knowledge, the first neutron structure of a carbohydrate-binding protein in complex with a fully deuterated carbohydrate ligand. A detailed structural analysis of the lectin-carbohydrate interactions provides information on the hydrogen bond network, the role of water molecules, and the extent of the CH-π stacking interactions between fucose and the aromatic amino acids in the binding site.

• Keywords
• Photorhabdus, carbohydrate-binding, carbohydrates, fully deuterated L-fucose, lectin, ligand binding, neutron macromolecular crystallography (NMX), neutron structure, perdeuteration, stacking interaction,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Fucose chemistry   metabolism   MeSH
• Lectins chemistry   metabolism   MeSH
• Photorhabdus chemistry   MeSH
• Protein Binding MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Fucose MeSH
• Lectins MeSH
• Hydrogen MeSH

Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria - BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed β-propeller fold. Functional studies conducted on a series of oligo- and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-l-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

• Keywords
• Burkholderia pseudomallei, Chromobacterium violaceum, Lectin, Protein structure, Seven-bladed β-propeller fold,
• MeSH
• Bacterial Proteins metabolism   MeSH
• Burkholderia pseudomallei metabolism   MeSH
• Chromobacterium metabolism   MeSH
• Fucose metabolism   MeSH
• Lectins metabolism   MeSH
• Humans MeSH
• Monosaccharides metabolism   MeSH
• Polysaccharides metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Fucose MeSH
• Lectins MeSH
• Monosaccharides MeSH
• Polysaccharides MeSH

Lectins with a β-propeller fold bind glycans on the cell surface through multivalent binding sites and appropriate directionality. These proteins are formed by repeats of short domains, raising questions about evolutionary duplication. However, these repeats are difficult to detect in translated genomes and seldom correctly annotated in sequence databases. To address these issues, we defined the blade signature of the five types of β-propellers using 3D-structural data. With these templates, we predicted 3,887 β-propeller lectins in 1,889 species and organized this information in a searchable online database. The data reveal a widespread distribution of β-propeller lectins across species. Prediction also emphasizes multiple architectures and led to the discovery of a β-propeller assembly scenario. This was confirmed by producing and characterizing a predicted protein coded in the genome of Kordia zhangzhouensis. The crystal structure uncovers an intermediate in the evolution of β-propeller assembly and demonstrates the power of our tools.

• Keywords
• carbohydrate binding protein, lectins, oligomerization, β-propeller,
• MeSH
• Archaea chemistry   MeSH
• Bacteria chemistry   MeSH
• Databases, Protein MeSH
• Eukaryota chemistry   MeSH
• Genome, Bacterial MeSH
• Lectins chemistry   MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Proteome MeSH
• Protein Folding MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lectins MeSH
• Proteome MeSH

The Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications.

• Keywords
• Aspergillus fumigatus, SPR, lectin, pathogen, protein–saccharide complex,
• MeSH
• Aspergillus fumigatus chemistry   MeSH
• Epithelium MeSH
• Fungal Proteins chemistry   MeSH
• Lectins chemistry   MeSH
• Humans MeSH
• Oligosaccharides chemistry   MeSH
• Protein Structure, Tertiary MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fungal Proteins MeSH
• Lectins MeSH
• Oligosaccharides MeSH

Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, Le(Y) being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,L-selenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus' conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection.

• MeSH
• Aspergillus fumigatus chemistry   MeSH
• Aspergillosis immunology   MeSH
• Bronchi cytology   microbiology   MeSH
• Epitopes chemistry   MeSH
• Virulence Factors chemistry   MeSH
• Fucose chemistry   MeSH
• Galactose chemistry   MeSH
• Genome, Fungal MeSH
• Hemagglutination MeSH
• Host-Pathogen Interactions MeSH
• Interleukin-8 metabolism   MeSH
• N-Acetylneuraminic Acid chemistry   MeSH
• Lectins chemistry   MeSH
• Humans MeSH
• Mannose chemistry   MeSH
• Molecular Sequence Data MeSH
• Oligosaccharides chemistry   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Sequence Alignment MeSH
• Spores, Fungal chemistry   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Epitopes MeSH
• Virulence Factors MeSH
• fucose-binding lectin MeSH Browser
• Fucose MeSH
• Galactose MeSH
• Interleukin-8 MeSH
• N-Acetylneuraminic Acid MeSH
• Lectins MeSH
• Mannose MeSH
• Oligosaccharides MeSH

The opportunistic pathogen Burkholderia cenocepacia expresses several soluble lectins, among them BC2L-C. This lectin exhibits two domains: a C-terminal domain with high sequence similarity to the recently described calcium-dependent mannose-binding lectin BC2L-A, and an N-terminal domain of 156 amino acids without similarity to any known protein. The recombinant N-terminal BC2L-C domain is a new lectin with specificity for fucosylated human histo-blood group epitopes H-type 1, Lewis b, and Lewis Y, as determined by glycan array and isothermal titration calorimetry. Methylselenofucoside was used as ligand to solve the crystal structure of the N-terminal BC2L-C domain. Additional molecular modeling studies rationalized the preference for Lewis epitopes. The structure reveals a trimeric jellyroll arrangement with striking similarity to TNF-like proteins, and to BclA, the spore protein from Bacillus anthracis which may play an important role in bioadhesion of anthrax spores in human lungs.

• MeSH
• Blood Group Antigens chemistry   immunology   metabolism   MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Burkholderia chemistry   metabolism   MeSH
• Epitopes chemistry   immunology   metabolism   MeSH
• Fucose chemistry   metabolism   MeSH
• Protein Structure, Quaternary MeSH
• Lectins chemistry   metabolism   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Blood Group Antigens MeSH
• Bacterial Proteins MeSH
• Epitopes MeSH
• Fucose MeSH
• Lectins MeSH

We present the results of a series of 10-ns molecular dynamics simulations on Pseudomonas aeruginosa lectin-II (PA-IIL) and its complexes with four different monosaccharides. We compare the saccharide-free, saccharide-occupied, and saccharide- and ion-free forms of the lectin. The results are coupled with analysis of the water density map and calcium coordination. The water density pattern around the binding site in the free lectin molecular dynamics was fitted with that in the X-ray and with the hydroxyl groups of the monosaccharide within the lectin/monosaccharide complexes and the best ligand was predicted based on the best fit. Interestingly, the water density pattern around the binding site in the uncomplexed lectin exactly fitted the O2, O3, and O4 hydroxyl groups of the fucose complex with the lectin. This observation could lead to a hypothesis that the replacement of these three water molecules from the binding site by the monosaccharide decreases the entropy of the complex and increases the entropy of the water molecules, which favors the binding. It suggests that the high density peaks of the solvent around the binding site in the free protein could be the tool to predict hydroxyl group orientation of the sugar in the protein/sugar complexes. The high affinity of PA-IIL binding site is also attributed to the presence of two calcium ions, each of them making five to six coordinations with the protein part and two coordinations with either water or the monosaccharide. When the calcium ions are removed from the simulated system, they are replaced by sodium ions from the solvent. These observations rationalize the high binding affinity of PA-IIL towards fucose.

• MeSH
• Carbohydrate Conformation MeSH
• Oxygen chemistry   MeSH
• Lectins chemistry   MeSH
• Models, Molecular * MeSH
• Monosaccharides chemistry   MeSH
• Computer Simulation MeSH
• Pseudomonas aeruginosa chemistry   MeSH
• Protein Structure, Secondary MeSH
• Temperature MeSH
• Calcium MeSH
• Binding Sites MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oxygen MeSH
• Lectins MeSH
• Monosaccharides MeSH
• Calcium MeSH
• Water MeSH

BACKGROUND: Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose--a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. RESULTS: In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22-23-24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions--both of afore mentioned have strong effects on the saccharide preferences. CONCLUSION: Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.

• MeSH
• Adhesins, Bacterial chemistry   genetics   MeSH
• Chromatography, Affinity MeSH
• Polymorphism, Single Nucleotide MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Lectins chemistry   genetics   MeSH
• Models, Molecular MeSH
• Monosaccharides chemistry   MeSH
• Mutagenesis, Site-Directed MeSH
• Protein Engineering MeSH
• Pseudomonas aeruginosa genetics   MeSH
• Ralstonia solanacearum chemistry   MeSH
• Recombinant Proteins chemistry   isolation & purification   MeSH
• Plant Lectins chemistry   MeSH
• Amino Acid Substitution MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adhesin, Pseudomonas MeSH Browser
• Adhesins, Bacterial MeSH
• Lectins MeSH
• Monosaccharides MeSH
• Recombinant Proteins MeSH
• Plant Lectins MeSH

The purple pigmented bacterium Chromobacterium violaceum is a dominant component of tropical soil microbiota that can cause rare but fatal septicaemia in humans. Its sequenced genome provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications and allowed an ORF encoding a protein that is 60% identical to the fucose binding lectin (PA-IIL) from Pseudomonas aeruginosa and the mannose binding lectin (RS-IIL) from Ralstonia solanacearum to be identified. The lectin, CV-IIL, has recently been purified from C. violaceum [Zinger-Yosovich, K., Sudakevitz, D., Imberty, A., Garber, N. C., and Gilboa-Garber, N. (2006) Microbiology 152, 457-463] and has been confirmed to be a tetramer with subunit size of 11.86 kDa and a binding preference for fucose. We describe here the cloning of CV-IIL and its expression as a recombinant protein. A complete structure-function characterization has been made in an effort to analyze the specificity and affinity of CV-IIL for fucose and mannose. Crystal structures of CV-IIL complexes with monosaccharides have yielded the molecular basis of the specificity. Each monomer contains two close calcium cations that mediate the binding of the monosaccharides, which occurs in different orientations for fucose and mannose. The thermodynamics of binding has been analyzed by titration microcalorimetry, giving dissociation constants of 1.7 and 19 microM for alpha-methyl fucoside and alpha-methyl mannoside, respectively. Further analysis demonstrated a strongly favorable entropy term that is unusual in carbohydrate binding. A comparison with both PA-IIL and RS-IIL, which have binding preferences for fucose and mannose, respectively, yielded insights into the monosaccharide specificity of this important class of soluble bacterial lectins.

• MeSH
• Bacterial Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Chromobacterium chemistry   metabolism   MeSH
• Entropy MeSH
• Fucose metabolism   MeSH
• Crystallization MeSH
• Mannose-Binding Lectin chemistry   genetics   isolation & purification   metabolism   MeSH
• Lectins chemistry   genetics   isolation & purification   metabolism   MeSH
• Mannose metabolism   MeSH
• Models, Molecular MeSH
• Recombinant Proteins metabolism   MeSH
• Solubility MeSH
• Protein Structure, Secondary MeSH
• Sensitivity and Specificity MeSH
• Static Electricity MeSH
• Calcium chemistry   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Hydrogen Bonding MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Bacterial Proteins MeSH
• fucose-binding lectin MeSH Browser
• Fucose MeSH
• Mannose-Binding Lectin MeSH
• Lectins MeSH
• Mannose MeSH
• Recombinant Proteins MeSH
• Calcium MeSH