Investigation of the Molecular Details of the Interactions of Selenoglycosides and Human Galectin-3.

Human galectin-3 (hGal-3) is involved in a variety of biological processes and is implicated in wide range of diseases. As a result, targeting hGal-3 for clinical applications has become an intense area of research. As a step towards the development of novel hGal-3 inhibitors, we describe a study of the binding of two Se-containing hGal-3 inhibitors, specifically that of di(ß-D-galactopyranosyl)selenide (SeDG), in which two galactose rings are linked by one Se atom and a di(ß-D-galactopyranosyl)diselenide (DSeDG) analogue with a diseleno bond between the two sugar units. The binding affinities of these derivatives to hGal-3 were determined by 15N-1H HSQC NMR spectroscopy and fluorescence anisotropy titrations in solution, indicating a slight decrease in the strength of interaction for SeDG compared to thiodigalactoside (TDG), a well-known inhibitor of hGal-3, while DSeDG displayed a much weaker interaction strength. NMR and FA measurements showed that both seleno derivatives bind to the canonical S face site of hGal-3 and stack against the conserved W181 residue also confirmed by X-ray crystallography, revealing canonical properties of the interaction. The interaction with DSeDG revealed two distinct binding modes in the crystal structure which are in fast exchange on the NMR time scale in solution, explaining a weaker interaction with hGal-3 than SeDG. Using molecular dynamics simulations, we have found that energetic contributions to the binding enthalpies mainly differ in the electrostatic interactions and in polar solvation terms and are responsible for weaker binding of DSeDG compared to SeDG. Selenium-containing carbohydrate inhibitors of hGal-3 showing canonical binding modes offer the potential of becoming novel hydrolytically stable scaffolds for a new class of hGal-3 inhibitors.

Binding of Starch Fragments to the Starch Branching Enzyme: Implications for Developing Slower-Digesting Starch.

Molecular weight distributions of starch branches affect functional properties, which can be controlled by engineering starch branching enzymes (SBEs). Molecular dynamics and docking approaches are used to examine interactions between SBE and starch fragments. In the native protein, three residues formed stable interactions with starch fragments in the central binding region; these residues may play key roles in substrate recognition. Fragments containing 5-12 glucose units interacted more tightly with SBE than smaller fragments, suggesting a minimal functional fragment size of 5, agreeing with experiment. Effects of three different point mutations on interactions with maltopentaose in the central binding region correlated well with experiment. Simulations indicate that SBE may template formation of the crystalline lamellae characteristic of native starch, consistent with the observation that crystalline lamellae formed by starch in a plant, are not necessarily the state of lowest free energy. The methodology will help develop starches with optimized functional properties.

Structure-Based Design of a Monosaccharide Ligand Targeting Galectin-8.

Galectin-8 is a ß-galactoside-recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3-O-[1-carboxyethyl]-ß-d-galactopyranoside (compound 6) as a ligand to target the N-terminal domain of galectin-8 (galectin-8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3'-O-sialylated lactose (3'-SiaLac), with galectin-8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin-8N for 6 was 32.8 µm, whereas no significant affinity was detected for the C-terminal domain of galectin-8 (galectin-8C). The crystal structure of the galectin-8N-6 complex validated the predicted binding conformation and revealed the exact protein-ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin-8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide-based scaffold as a binder of galectin-8.

The characterization of modified starch branching enzymes: toward the control of starch chain-length distributions.

Starch is a complex branched glucose polymer whose branch molecular weight distribution (the chain-length distribution, CLD) influences nutritionally important properties such as digestion rate. Chain-stopping in starch biosynthesis is by starch branching enzyme (SBE). Site-directed mutagenesis was used to modify SBEIIa from Zea mays (mSBEIIa) to produce mutants, each differing in a single conserved amino-acid residue. Products at different times from in vitro branching were debranched and the time evolution of the CLD measured by size-exclusion chromatography. The results confirm that Tyr352, Glu513, and Ser349 are important for mSBEIIa activity while Arg456 is important for determining the position at which the linear glucan is cut. The mutant mSBEIIa enzymes have different activities and suggest the length of the transferred chain can be varied by mutation. The work shows analysis of the molecular weight distribution can yield information regarding the enzyme branching sites useful for development of plants yielding starch with improved functionality.