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. 2018 Aug 17;13(8):2211-2219.
doi: 10.1021/acschembio.8b00377. Epub 2018 Jun 12.

Effect of Noncanonical Amino Acids on Protein-Carbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered With Fluorinated Tryptophan Analogs

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Effect of Noncanonical Amino Acids on Protein-Carbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered With Fluorinated Tryptophan Analogs

Felix Tobola et al. ACS Chem Biol. .
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Abstract

Protein-carbohydrate 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 Ralstonia solanacearum. 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 carbohydrate-aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(a) Structure of RSL (pdb 2BT9). The three monomers are colored in magenta, green, and cyan; the bound αMeFuc is represented as spheres. (b) The intermonomeric binding site with three important Trp residues: W31, W36, and W53 (structurally equivalent to W76, W81, and W10 in the intramonomeric site). (c) Structures of l-tryptophan and the fluorinated l-analogs used in this study.
Figure 2
Figure 2
ITC experiment of RSL and fluorinated RSL binding to the tetrasaccharide LeX. (a) Thermogram (top) of the injection of LeX aliquots to RSL in solution and corresponding integrated peaks (bottom) for RSL and the variants. (b) The thermodynamic contributions were derived from curve fitting. Entropy cost, −TΔS; enthalpy contribution, ΔH; free energy of binding, ΔG.
Figure 3
Figure 3
(a) Superimposition of the crystal structure of the RSL/αMeFuc complex (white; pdb 2BT9) with the structures of RSL[4FW] (yellow; pdb 5O7W), RSL[5FW] (cyan; pdb 5O7V), and RSL[7FW] (green; pdb 5O7U). (b) Superimposition of loop 76–82 from wt-RSL (white), and chain A (yellow) and chain B (orange) of RSL[4FW]. Fluorine atoms are colored in purple.
Figure 4
Figure 4
(a) Superimposition of the six intermonomeric binding sites (chains A and B of the three RSL variants) complexed with the LeX tetrasaccharide. Color coding as in Figure 3a. (b) Close-up of the intermonomeric binding site of RSL[7FW] (chain B). The network of hydrogen bonds is displayed as dashed blue lines, and contacts with the fluorine atoms are in magenta.
Figure 5
Figure 5
Instability of the hydrogen bond network involving the Fuc-HO4 hydroxyl group in RSL[7FW]. History of distances for competing hydrogen bonds between HO4 and either Trp36.F or Glu73.OE1, and snapshots demonstrating the occurrence of different orientations of fucose in the binding site, which results in alternative hydrogen bond networks. Arrows indicate the position of the two snapshots in the trajectories.
Figure 6
Figure 6
(a) Comparison of the blood group oligosaccharide-binding specificity of RSL and RSL[7FW]. Full names and chemical structures of the glycans are shown in Figure S11. (b) Docking of blood group B trisaccharide into the intermonomeric site of RSL[7FW] reveals nonfavorable short contacts between the fluorine atoms at Trp53 and Trp31 and the CH groups at positions C3 and C4 of galactose.

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