Stacking Interactions between Carbohydrate and Protein Quantified by Combination of Theoretical and Experimental Methods

<div><p>Carbohydrate – receptor interactions are an integral part of biological events. They play an important role in many cellular processes, such as cell-cell adhesion, cell differentiation and in-cell signaling. Carbohydrates can interact with a receptor by using several types of intermolecular interactions. One of the most important is the interaction of a carbohydrate's apolar part with aromatic amino acid residues, known as dispersion interaction or CH/π interaction. In the study presented here, we attempted for the first time to quantify how the CH/π interaction contributes to a more general carbohydrate - protein interaction. We used a combined experimental approach, creating single and double point mutants with high level computational methods, and applied both to <em>Ralstonia solanacearum</em> (RSL) lectin complexes with α-l-Me-fucoside. Experimentally measured binding affinities were compared with computed carbohydrate-aromatic amino acid residue interaction energies. Experimental binding affinities for the RSL wild type, phenylalanine and alanine mutants were −8.5, −7.1 and −4.1 kcal.mol⁻¹, respectively. These affinities agree with the computed dispersion interaction energy between carbohydrate and aromatic amino acid residues for RSL wild type and phenylalanine, with values −8.8, −7.9 kcal.mol⁻¹, excluding the alanine mutant where the interaction energy was −0.9 kcal.mol⁻¹. Molecular dynamics simulations show that discrepancy can be caused by creation of a new hydrogen bond between the α-l-Me-fucoside and RSL. Observed results suggest that in this and similar cases the carbohydrate-receptor interaction can be driven mainly by a dispersion interaction.</p> </div

Sequence alignment of PHL (NCBI Reference Sequence: WP_012776886.1) and PLL (Sequence ID: 5C9L_A) proteins.

<p>The asterisk indicates fully conserved residues, the colon a strong conservation between groups of amino acids and the dot is for weak similarity.</p

Comparison of PHL/Me-α-l-Fuc and PLL/l-Fuc (PDB ID: 5C9P) structure.

<p>Top (A) and bottom (B) view of PHL (yellow/green) and PLL (magenta/pink) monomer. Differences are mainly visible in loop regions. (C) Sequence alignment of PHL/PLL repetitions. Residues responsible for l-Fuc or d-Gal binding are highlighted with a magenta and orange background, respectively. Different residues in the binding sites shown in bold.</p

Data collection and refinement statistics.

<p>Values in parentheses correspond to highest resolution shell.</p

Haemagglutination inhibition assay with PHL using microscopy.

<p>Minimal inhibitory concentrations (MIC) of carbohydrate ligands and their potency towards l-fucose were determined.</p

Box and whisker plot for glycan array screening for PHL (200 μg/ml) labeled with DyLight 488 NHS Ester.

<p>The top 19 saccharides and control trehalose (sample 20) were selected for display. The 7 saccharides giving the highest average signals are depicted. Linker formula—sp2: -O-CH₂CH₂NH₂; sp3: -O-(CH₂)₃NH₂. The bottom and top of the box are the first and third quartiles; the band inside the box is the second quartile (the median); the ends of the whiskers represent the minimum and maximum values of the data and the small squares inside the boxes represent the mean. RFU, relative fluorescence units. Complete glycan array results and the raw data are given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006564#ppat.1006564.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006564#ppat.1006564.s002" target="_blank">S2</a> Tables, respectively.</p

Localization of Fuc- and Gal-specific sites with respect to the PHL dimer.

<p>Residues closer than 4Å from the observed ligands are coloured.</p

Determination of carbohydrate specificity/affinity of PHL towards l-fucose and d-galactose.

<p><b>(A)</b> SPR sensorgrams (differential curves) displaying PHL binding (0.25 μM) to CM5 sensor chip with immobilized l-fucoside in the presence of competing saccharides. Response decreases with increasing concentration of the inhibitor–l-fucose (0–50 mM), d-galactose (0–250 mM). <b>(B)</b> A logarithmic plot of inhibition curves calculated from SPR measurements <b>(C)</b> ITC curves of PHL (50 μM) titration by l-fucose (20 mM) and d-galactose (50 mM). 20 injections of 2.0 μl of sugars were added every 240 s to a PHL-containing cell. Lower plots show the total heat released as a function of total ligand concentration for the titration shown in the upper panels.</p

Equilibrium dissociation binding constants for interaction between PHL and carbohydrate ligands determined by isothermal titration calorimetry at 25°C (standard deviations were calculated from three independent measurements).

<p>Equilibrium dissociation binding constants for interaction between PHL and carbohydrate ligands determined by isothermal titration calorimetry at 25°C (standard deviations were calculated from three independent measurements).</p

Phenoloxidase (PO) activity in haemolymph of <i>G</i>. <i>mellonella</i>.

<p>(A) Haemolymph samples were incubated with PBS or different amounts of PHL and the melanisation catalysed by PO was measured at 492 nm after mixing with 3,4-dihydroxy-dl-phenylalanine substrate solution. (B) Haemolymph was incubated with PBS, BSA (475 μg), PLL (475 μg) or PHL (475 μg; active or inhibited by 0.2M l-Fuc and Me-α-l-Fuc) and subsequently mixed with the substrate 3,4-dihydroxy-dl-phenylalanine. Products of melanisation catalysed by PO were measured at 492 nm. Data presented as means ± SD; * indicates significant difference p < 0.05, ** p < 0.01 (Dunnett's and Tukey’s test, respectively).</p