Unravelling the Time Scale of Conformational Plasticity and Allostery in Glycan Recognition by Human Galectin‐1

Abstract

The interaction of human galectin‐1 with a variety of oligosaccharides, from di‐(N‐acetyllactosamine) to tetra‐saccharides (blood B type‐II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand‐ and receptor‐based NMR experiments assisted by computational methods allowed proposing three‐dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein‐sugar interactions. CLEANEX‐PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond‐millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin‐1.

Exploration of Galectin Ligands Displayed on Gram-Negative Respiratory Bacterial Pathogens with Different Cell Surface Architectures

“…Human Gal-1 (unlabelled) and N -terminally His-tagged human Gal-3, Gal-4, and Gal-8 were obtained from ATGen Ltd. A second preparation of recombinant human Gal-1 was produced in BL21 E. coli cells via induction with isopropyl β-D-1-thio-galactopyranoside [ 21 ]. Following cell lysis, Gal-1 was purified by affinity chromatography on lactose-agarose (Sigma-Aldrich), using lactose-containing buffer as eluant.…”

“…Following cell lysis, Gal-1 was purified by affinity chromatography on lactose-agarose (Sigma-Aldrich), using lactose-containing buffer as eluant. After extensive dialysis of the eluted fraction, Gal-1 purity was checked by SDS-PAGE and LC-MS, and the absence of lactose was confirmed by NMR spectroscopy [ 21 ].…”

The two domains of human galectin-8 bind sialyl- and fucose-containing oligosaccharides in an independent manner. A 3D view by using NMR

“…The N-terminal CRD showed a larger number of highly dynamic interactions with the peptide linker. However, and applying the methodology previously reported by our group to analyse allosteric networks, 30 it was observed that the correlated motion of binding residues quickly dissipated nearby each binding sites without affecting the interdomain peptide (Fig. S14, ESI † ).…”

Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers

“…Fittingly, the CSP plots obtained for Gal-1 and Gal-3 in the presence of 1 correspond to the profile for these interactors (Figure 3). Only the canonical binding site of the lectin was perturbed (mainly strands S5 and S6) [8,17,18]. Interestingly, a different behavior was observed for the interactions with ligands 2 and 3: A significant reduction of the intensities of the 1 H- 15 N HSQC cross-peaks of both lectins was detected during these titrations, especially when Gal-1 was employed.…”

“…In this work, we employed ligand-based (STD-NMR) and receptor-based ( 1 H- 15 N HSQC) NMR methods to examine, with atomic resolution, the intermolecular interaction of Gal-1 and Gal-3 CRD with a series of five multivalent compounds that display different structural scaffolds. The ligands tested as putative inhibitors of galectin-mediated interactions presented a common LacNAc (Galβ4GlcNAc) motif (Figure 1) [17][18][19]. STD-NMR experiments provide information on the existence of interaction and on the precise ligand binding epitope, while 1 H- 15 N HSQC spectra make it possible to assess the interaction, as well as deduce the protein binding site, the existence of exchange phenomena, and the possible generation of larger-order complexes.…”