Surface representation of Gal-8C domain complexed with di-LacNAc.

<p>Carbohydrates are shown in stick. The ligands are color-coded (β-galactose: red; N-acetyl-glucosamine: green; and downstream hydroxy group: white. (<b>A</b>) Interaction of terminal β-galactose of di-LacNAc. (<b>B</b>) Interaction of internal β-galactose di-LacNAc.</p

Torsional analysis of bound ligands.

<p>Average glycosidic torsion angles for bound ligands in the Gal-8C domain (standard deviation). φ and ψ values for glycosidic linkages using the NMR definition as H1-C1-O1-C_x and C1-O1-C_x-H_x respectively.</p

Structure superimposition and degree of sequence identity.

<p>Three-dimensional structural alignments and sequence identity of members of the galectin family based on RMSD calculated by using the PDBeFold webserver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059761#pone.0059761-Krissinel1" target="_blank">[49]</a>.</p

MM/GBSA energies.

<p>All values are reported in kcal/mol. ΔE_elec, electrostatic molecular mechanical energy; ΔE_vdw, van der Walls molecular mechanical energy; ΔE_MM = ΔE_elec+ΔE_vdw, total molecular mechanical energy; ΔG_np, non-polar contribution to the solvation energy; ΔG_p, polar contribution to the solvation energy; ΔG_solv = ΔG_np+ΔG_p, total solvation energy; ΔG_total, total energy (without entropy contribution); –ΤΔS, -T (temperature)*ΔS(sum of rotational, translational and vibrational entropies); ΔG_binding total binding energy of the system.</p

Superimposition of Gal-8N and -C domain.

<p>Ribbon representation of superimposed Gal-8N and -C domain. The N domain is shown in pink color code whereas the C domain is in cyan. Lactose is shown as stick model in yellow color. The variable loop between S3–S4 shows difference in length between Gal-8C and -N.</p

Multiple sequence alignments of the human galectin members.

<p>Conserved amino acids are shown in bold, amino acids which play important roles in interactions apart from conserved residues in Gal-8C are shown in red and in blue for Gal-8N. This multiple sequence alignment was carried out by MAFFT web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059761#pone.0059761-Katoh1" target="_blank">[48]</a>.</p

Understanding the Specificity of Human Galectin-8C Domain Interactions with Its Glycan Ligands Based on Molecular Dynamics Simulations

<div><p>Human Galectin-8 (Gal-8) is a member of the galectin family which shares an affinity for β-galactosides. The tandem-repeat Gal-8 consists of a N- and a C-terminal carbohydrate recognition domain (N- and C-CRD) joined by a linker peptide of various length. Despite their structural similarity both CRDs recognize different oligosaccharides. While the molecular requirements of the N-CRD for high binding affinity to sulfated and sialylated glycans have recently been elucidated by crystallographic studies of complexes with several oligosaccharides, the binding specificities of the C-CRD for a different set of oligosaccharides, as derived from experimental data, has only been explained in terms of the three-dimensional structure for the complex C-CRD with lactose. In this study we performed molecular dynamics (MD) simulations using the recently released crystal structure of the Gal-8C-CRD to analyse the three-dimensional conditions for its specific binding to a variety of oligosaccharides as previously defined by glycan-microarray analysis. The terminal β-galactose of disaccharides (LacNAc, lacto-N-biose and lactose) and the internal β-galactose moiety of blood group antigens A and B (BGA, BGB) as well as of longer linear oligosaccharide chains (di-LacNAc and lacto-N-neotetraose) are interacting favorably with conserved amino acids (H53, R57, N66, W73, E76). Lacto-N-neotetraose and di-LacNAc as well as BGA and BGB are well accommodated. BGA and BGB showed higher affinity than LacNAc and lactose due to generally stronger hydrogen bond interactions and water mediated hydrogen bonds with α1-2 fucose respectively. Our results derived from molecular dynamics simulations are able to explain the glycan binding specificities of the Gal-8C-CRD in comparison to those of the Gal-8N -CRD.</p> </div

Set of oligosaccharide ligands.

<p>List of oligosaccharides used in MD simulations for study of interactions with the Gal-8C domain.</p

Ligand binding of the galectin-8C domain.

<p>The Gal-8C binding site with (<b>A</b>) LacNAc, II, (<b>B</b>) di-LacNAc, (<b>C</b>) Lactose, (<b>D</b>) Lacto-N-neotetrose, (<b>E</b>) BGA, and (<b>F</b>) BGB. Ligands are shown as stick models and the surface of the protein-binding site in violet color. The ligands are color-coded (β-galactose: red; N-acetyl-glucosamine: green; glucose: blue; fucose: cyan; α-galactose and α- N-acetyl-galactosamine: yellow; downstream hydroxy group: white. Hydrogen bonds are shown as yellow dotted line. A snapshot which contains a maximum number of intermolecular hydrogen bonds is displayed. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059761#pone.0059761.s007" target="_blank"><b>File S1</b></a> for details of hydrogen bond interactions of each complex. The figure was designed using PyMOL Molecular Graphics System (DeLano Scientific, Palo Alto, CA).</p

Oligosaccharides ranked by calculated binding energy towards the Gal-8C domain.

<p>The values are derived from MMGBSA energies and entropy values calculated using NMode.</p