NMR-Based, Molecular Dynamics- and Random Walk Molecular Mechanics-Supported Study of Conformational Aspects of a Carbohydrate Ligand (Galβ1-2Galβ1-R) for an Animal Galectin in the Free and in the Bound State

Abstract

The binding of a carbohydrate to a lectin may affect the conformation of the ligand. To address this question for the galectin from chicken liver, the conformation of Gal␤1-2Gal␤1-R was analyzed in the free and in the galectin-bound state with 2D-ROESY-and 1D-as well as 2D-transferred NOE-experiments. A computerassisted analysis of spatial parameters of the ligand by molecular dynamics (MD) and random walk molecular mechanics (RAMM) calculations, taking different dielectric constants from ⑀ ‫ס‬ 1 to ⑀ ‫ס‬ 80 and various force fields into account, were instrumental to define the energetic minima of the free state. NMR-derived interresidual distance constraints enabled a conformational mapping. The two overlapping interresidual distance constraints obtained from transferred-NOE experiments of the galectin-ligand complex clearly support the notion that the conformation of the disaccharide in the bound state is at least very close to its global energy minimum state in solution. © 1996 Academic Press, Inc.Protein-carbohydrate interactions can guide crucial physiological reactions such as cell adhesion, inter-and intracellular glycoprotein routing and regulation of various cellular functions (1, 2). Their evident importance prompts to analyze in detail the molecular characteristics of this type of recognition in solution. The combination of computer-assisted calculations of the conformation and NMR-based experiments for the free ligand and lectin-saccharide complex provides a suitable tool to address this question. It enables us to compare spatial parameters of a certain ligand before and after association with members of a lectin family with similar binding specificity, e.g. to galactosides. Agglutinins of this class mediate cell-matrix interactions and enhance immune functions (3-5). Due to the remarkable affinity of Gal␤1-2Gal␤1-R to the immunomodulatory Viscum album agglutinin, reported previously (6, 7), we have chosen to map conformational aspects of this high-affinity ligand for galactoside-binding lectins of organisms from different branches of the evolutionary tree. Herein, we report the first study in this area for an animal galectin. Transferred nuclear Overhauser effect (TRNOE)-based experiments in combination with conformational distance mapping, random walk molecular mechanics (RAMM) and molecular dynamics (MD) calculations indicate that the actual conformations of this disaccharide in the complex and in the free form are at least very similar.

Involvement of Laser Photo‐CIDNP(Chemically Induced Dynamic Nuclear Polarization)‐Reactive Amino Acid Side Chains in Ligand Binding by Galactoside‐Specific Lectins in Solution

“…To understand in detail the underlying relay system analysis of crystals of mutant proteins will be conducive. Supplementary information on ligand-binding characteristics in solution can be gained by transferred NOE studies of complexes and by application of custom-made carbohydrate derivatives with fluoro or deoxy substitutions (Siebert et al, 1996b(Siebert et al, , 1997bSolis et al, 1996;Solis and Diaz-MauriAo, 1997). By using both these ligand site-directed approaches and a spectroscopic method such as laser photo-CIDNP measurements the extent of experimental evidence increases the reliability of answers to the problems, explicitly given in this report.…”

Activity–structure correlations in divergent lectin evolution: fine specificity of chicken galectin CG-14 and computational analysis of flexible ligand docking for CG-14 and the closely related CG-16

“…These rather rigid ligands ( please see Figure 5 for accessible conformational space) appear to home into the CRDs without a major entropic penalty in the thermodynamic balance sheet owing to the loss of conformational freedom. The implied selection of a low-energy conformer present in solution has already been described for CG-16 and the disaccharide Galb1-2Gal (Siebert et al 1996). The computed topology of the interactions between the tri-and tetrasaccharides and the CRDs provide a first view on the extended binding site of these animal lectins for a1-2/a1-3-substituted b-galactosides.…”

Knowledge-based Homology Modeling and Experimental Determination of Amino Acid Side Chain Accessibility by the Laser Photo CIDNP (Chemically Induced Dynamic Nuclear Polarization) Approach in Solution: Lessons from the Small Sialidase of Clostridium perfringens

“…dumping hydrogen bond forces), as already shown e.g. in the cases of the oligosaccharide chain of the ganglioside 9-O-acetyl-GD1a and a galectin-binding disaccharide (Galb1-2Galb1-R) [31][32][33][34][35]. A production period of 100 ps and an integration step of 1 fs was necessary to produce reliable trajectories within each simulation.…”

“…Intriguingly, the changes in surface accessibilities of widely separated residues affected by a Tyr/ Phe-or a Cys/Ser-substitution intimate that disparate conformational changes of mutant enzymes relative to the wildtype form should not be underestimated. Extending the combination of computational and NMR techniques, as illustrated in recent reports and reviews with emphasis on sugar-binding proteins [31][32][33][34][35], it will technically amenable to figure out reliably, whether such rather global alterations can affect carbohydrate binding properties. …”