34 research outputs found
The Hidden Conformation of Lewis x, a Human Histo-Blood Group Antigen, Is a Determinant for Recognition by Pathogen Lectins
Histo-blood group epitopes are fucosylated
branched oligosaccharides
with well-defined conformations in solution that are recognized by
receptors, such as lectins from pathogens. We report here the results
of a series of experimental and computational endeavors revealing
the unusual distortion of histo-blood group antigens by bacterial
and fungal lectins. The Lewis x trisaccharide adopts a rigid closed
conformation in solution, while crystallography and molecular dynamics
reveal several higher energy open conformations when bound to the <i>Ralstonia solanacearum</i> lectin, which is in agreement with
thermodynamic and kinetic measurements. Extensive molecular dynamics
simulations confirm rare transient Le<sup>x</sup> openings in solution,
frequently assisted by distortion of the central N-acetyl-glucosamine
ring. Additional directed molecular dynamic trajectories revealed
the role of a conserved tryptophan residue in guiding the fucose into
the binding site. Our findings show that conformational adaptation
of oligosaccharides is of paramount importance in cell recognition
and should be considered when designing anti-infective glyco-compounds
Multivalent Glycomimetics with Affinity and Selectivity toward Fucose-Binding Receptors from Emerging Pathogens
Bacterial
and fungal pathogens involved in lung infection in cystic
fibrosis patients utilize a particular family of glycan-binding proteins,
characterized by the presentation of six fucose-binding sites on a
ring-shaped scaffold. These lectins are attractive targets for anti-infectious
compounds that could interfere in the recognition of host tissues
by pathogens. The design of a cyclopeptide-based hexavalent structure
allowed for the presentation of six fucose residues. The synthetic
hexavalent compound displays liable geometry resulting in high-avidity
binding by lectins from <i>Aspergillus fumigatus</i> and <i>Burkholderia ambifaria</i>. Replacing the
fucose residue with a conformationally constrained fucomimetic does
not alter the affinity and provides fine specificity with no binding
to other fucose-specific lectins
Effect of Noncanonical Amino Acids on ProteinâCarbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered with Fluorinated Tryptophan Analogs
Proteinâcarbohydrate
interactions play crucial roles in
biology. Understanding and modifying these interactions is of major
interest for fighting many diseases. We took a synthetic biology approach
and incorporated noncanonical amino acids into a bacterial lectin
to modulate its interactions with carbohydrates. We focused on tryptophan,
which is prevalent in carbohydrate binding sites. The exchange of
the tryptophan residues with analogs fluorinated at different positions
resulted in three distinctly fluorinated variants of the lectin from <i>Ralstonia solanacearum</i>. We observed differences in stability
and affinity toward fucosylated glycans and rationalized them by X-ray
and modeling studies. While fluorination decreased the aromaticity
of the indole ring and, therefore, the strength of carbohydrateâaromatic
interactions, additional weak hydrogen bonds were formed between fluorine
and the ligand hydroxyl groups. Our approach opens new possibilities
to engineer carbohydrate receptors
Discovery of Two Classes of Potent Glycomimetic Inhibitors of <i>Pseudomonas aeruginosa</i> LecB with Distinct Binding Modes
The
treatment of infections due to the opportunistic pathogen <i>Pseudomonas aeruginosa</i> is often difficult, as a consequence
of bacterial biofilm formation. Such a protective environment shields
the bacterium from host defense and antibiotic treatment and secures
its survival. One crucial factor for maintenance of the biofilm architecture
is the carbohydrate-binding lectin LecB. Here, we report the identification
of potent mannose-based LecB inhibitors from a screening of four series
of mannosides in a novel competitive binding assay for LecB. Cinnamide
and sulfonamide derivatives are inhibitors of bacterial adhesion with
up to a 20-fold increase in affinity to LecB compared to the natural
ligand methyl mannoside. Because many lectins of the host require
terminal saccharides (<i>e.g.</i>, fucosides), such capped
structures as reported here may offer a beneficial selectivity profile
for the pathogenic lectin. Both classes of compounds show distinct binding modes
at the protein, offering the advantage of a simultaneous development
of two new lead structures as anti-pseudomonadal drugs with an anti-virulence
mode of action
Energy maps and docked conformations.
<p>Isoenergy contrours (1 kcal/mole) are represented as a function of Ί (x-axis) and Κ (y-axis)torsion angles for all glycosidic linkages of interest taken from Glyco3D (<a href="http://glyco3d.cermav.cnrs.fr/" target="_blank">http://glyco3d.cermav.cnrs.fr/</a>) with superimposition of oligosaccharide conformations derived from docking (circles and squares) or protein crystallography (stars). Torsion angles are defined as Ίâ=âΞ(O5-C1-O1-Cx) and Κâ=âΞ(C1-O1-CâČx-CâČx+1).</p
Deciphering the Glycan Preference of Bacterial Lectins by Glycan Array and Molecular Docking with Validation by Microcalorimetry and Crystallography
<div><p>Recent advances in glycobiology revealed the essential role of lectins for deciphering the glycocode by specific recognition of carbohydrates. Integrated multiscale approaches are needed for characterizing lectin specificity: combining on one hand high-throughput analysis by glycan array experiments and systematic molecular docking of oligosaccharide libraries and on the other hand detailed analysis of the lectin/oligosaccharide interaction by x-ray crystallography, microcalorimetry and free energy calculations. The lectins LecB from <i>Pseudomonas aeruginosa</i> and BambL from <i>Burkholderia ambifaria</i> are part of the virulence factors used by the pathogenic bacteria to invade the targeted host. These two lectins are not related but both recognize fucosylated oligosaccharides such as the histo-blood group oligosaccharides of the ABH(O) and Lewis epitopes. The specificities were characterized using semi-quantitative data from glycan array and analyzed by molecular docking with the Glide software. Reliable prediction of protein/oligosaccharide structures could be obtained as validated by existing crystal structures of complexes. Additionally, the crystal structure of BambL/Lewis x was determined at 1.6 Ă
resolution, which confirms that Lewis x has to adopt a high-energy conformation so as to bind to this lectin. Free energies of binding were calculated using a procedure combining the Glide docking protocol followed by free energy rescoring with the Prime/Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method. The calculated data were in reasonable agreement with experimental free energies of binding obtained by titration microcalorimetry. The established predictive protocol is proposed to rationalize large sets of data such as glycan arrays and to help in lead discovery projects based on such high throughput technology.</p></div
Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity
Multivalency is proposed
to play a role in the strong avidity of
lectins for glycosylated cell surfaces and also in their ability to
affect membrane dynamics by clustering glycosphingolipids. Lectins
with modified valency were designed from the ÎČ-propeller fold
of Ralstonia solanacearum lectin (RSL)
that presents six fucose binding sites. After identification of key
amino acids by molecular dynamics calculations, two mutants with reduced
valency were produced. Isothermal titration calorimetry confirmed
the loss of three high affinity binding sites for both mutants. Crystal
structures indicated that residual low affinity binding occurred in
W76A but not in R17A. The trivalent R17A mutant presented unchanged
avidity toward fucosylated surfaces, when compared to hexavalent RSL.
However, R17A is not able anymore to induce formation of membrane
invaginations on giant unilamellar vesicules, indicating the crucial
role of number of binding sites for clustering of glycolipids. In
the human lung epithelial cell line H1299, wt-RSL is internalized
within seconds whereas the kinetics of R17A uptake is largely delayed.
Neolectins with tailored valency are promising tools to study membrane
dynamics
Flowchart of the docking/analysis protocol.
<p>Flowchart of the docking/analysis protocol.</p
Docking of the oligosaccharides in LecB binding site.
<p>a) H type 1, b) H type 2, c) Le<sup>a</sup>, d) Le<sup>x</sup>, e) sLe<sup>a</sup> f) sLe<sup>x</sup> and g) A-tri. The docked oligosaccharides are represented as sticks (carbon, oxygen and nitrogen atoms are colored green, red and blue respectively) and the ones from crystal structures are colored orange. Calcium ions are represented as pink spheres. The protein accessible surface is colored in beige for residues comprised within a sphere of 4 Ă
around the ligand and in blue for residues involved in hydrogen bond with ligand residues (except fucose).</p
Reduction of Lectin Valency Drastically Changes Glycolipid Dynamics in Membranes but Not Surface Avidity
Multivalency is proposed
to play a role in the strong avidity of
lectins for glycosylated cell surfaces and also in their ability to
affect membrane dynamics by clustering glycosphingolipids. Lectins
with modified valency were designed from the ÎČ-propeller fold
of Ralstonia solanacearum lectin (RSL)
that presents six fucose binding sites. After identification of key
amino acids by molecular dynamics calculations, two mutants with reduced
valency were produced. Isothermal titration calorimetry confirmed
the loss of three high affinity binding sites for both mutants. Crystal
structures indicated that residual low affinity binding occurred in
W76A but not in R17A. The trivalent R17A mutant presented unchanged
avidity toward fucosylated surfaces, when compared to hexavalent RSL.
However, R17A is not able anymore to induce formation of membrane
invaginations on giant unilamellar vesicules, indicating the crucial
role of number of binding sites for clustering of glycolipids. In
the human lung epithelial cell line H1299, wt-RSL is internalized
within seconds whereas the kinetics of R17A uptake is largely delayed.
Neolectins with tailored valency are promising tools to study membrane
dynamics