Structural characterisation of human galectin-4 N-terminal carbohydrate recognition domain in complex with glycerol, lactose, 3'-sulfo-lactose, and 2'-fucosyllactose

Abstract

Galectin-4 is a tandem-repeat galectin with two distinct carbohydrate recognition domains (CRD). Galectin-4 is expressed mainly in the alimentary tract and is proposed to function as a lipid raft and adherens junction stabilizer by its glycan cross-linking capacity. Galectin-4 plays divergent roles in cancer and inflammatory conditions, either promoting or inhibiting each disease progression, depending on the specific pathological condition. The study of galectin-4's ligand-binding profile may help decipher its roles under specific conditions. Here we present the X-ray structures of human galectin-4 N-terminal CRD (galectin-4N) bound to different saccharide ligands. Galectin-4's overall fold and its core interactions to lactose are similar to other galectin CRDs. Galectin-4N recognises the sulfate cap of 3'-sulfated glycans by a weak interaction through Arg45 and two water-mediated hydrogen bonds via Trp84 and Asn49. When galectin-4N interacts with the H-antigen mimic, 2'-fucosyllactose, an interaction is formed between the ring oxygen of fucose and Arg45. The extended binding site of galectin-4N may not be well suited to the A/B-antigen determinants, α-GalNAc/α-Gal, specifically due to clashes with residue Phe47. Overall, galectin-4N favours sulfated glycans whilst galectin-4C prefers blood group determinants. However, the two CRDs of galectin-4 can, to a less extent, recognise each other's ligands.

Figure 1. Structure of galectin-4N.

(A) Simulated annealing omit map of the calcium-binding site (engaging the S4–S5 loop) of the galectin-4N CRD with bound lactose. Galectin-4N CRD displayed as green cartoon representation with calcium- and lactose-binding site residues depicted in green sticks. Calcium ion is shown as a magenta sphere, its coordinating waters (red spheres) and the bound lactose molecule (stick model with carbon atoms in yellow). The S4–S5 loop cartoon and carbons are highlighted in lime green. Dashed lines highlight calcium coordination with its surroundings. The 2|Fo| − |Fc|α_calc map is illustrated in blue mesh at a 1.0 σ contour level and the |Fo| − |Fc|α_calc map is illustrated in red mesh at a 3.0 σ contour level. The omit map was created by Phenix. (B) Galectin-4N CRD shown in cartoon representation (β-strands (green), α-helical turn (red), loops (yellow)) with bound lactose molecule in stick representation (yellow carbons, red oxygens). The β-strands on the concave side of the β-sandwich and certain loops are labelled in black. Binding sub-sites are labelled in red. (C) Stereo image of galectin-4N CRD with side-chains of residues implicated in recognition of oligosaccharides displayed in stick representation (carbon (green) oxygen (red), nitrogen (blue)). Bound lactose molecule in transparent stick representation (carbon (yellow), oxygen (red)) indicates the conserved carbohydrate-binding site.

Figure 2. Crystal structure of galectin-4N-glycerol complex.

(A) Simulated annealing omit map of galectin-4N binding site with bound glycerol. Galectin-4N binding site residues (green carbons) with bound glycerol (carbon (yellow)) depicted in stick representation. The 2|Fo| − |Fc|α_calc map is illustrated in blue mesh at a 1.0 σ contour level and the |Fo| − |Fc|α_calc map is illustrated in red mesh at a 3.0 σ contour level. The omit map was created by the “composite omit map” task of Phenix. (B) Binding site interactions of galectin-4N and glycerol. Galectin-4N binding site depicted in green cartoon representation with binding site residues and bound glycerol (carbon (yellow)) represented as sticks. Hydrogen bonds are illustrated as dashed lines.

Figure 3. Crystal structure of galectin-4N-lactose complex.

(A) Simulated annealing omit map of galectin-4N binding site with bound lactose. Galectin-4N binding site residues (green carbons) with the bound lactose (yellow carbon) depicted in stick representation. The 2|Fo| – |Fc|α_calc map is illustrated in blue mesh at a 1.0 σ contour level and the |Fo| – |Fc|α_calc map is illustrated in red mesh at a 3.0 σ contour level. The omit map was created by the “composite omit map” task of Phenix. (B) Binding site interactions of galectin-4N and lactose. Galectin-4N binding site depicted in green cartoon representation with binding site residues (carbon (green)) and bound lactose (carbon (yellow)) represented as sticks. Hydrogen bonds are illustrated as dashes.

Figure 4. Crystal structure of galectin-4N-3′SuL complex.

(A) Simulated annealing omit map of galectin-4N binding site with bound 3′SuL. Galectin-4N binding site residues (carbon (green)) with the bound 3′SuL (carbon (yellow), sulphur (silver)) depicted in stick representation. The 2|Fo| – |Fc|α_calc map is illustrated in blue mesh at a 1.0 σ contour level and the |Fo| – |Fc|α_calc map is illustrated in red mesh at a 3.0 σ contour level. The omit map was created by the “composite omit map” task of Phenix. (B) Binding site interactions of galectin-4N and 3′SuL. Stereo image of galectin-4N binding site depicted in green cartoon representation with binding site residues (carbon (green); alternative conformations of Arg45 and Asp69 are illustrated with carbon (cyan)) and bound 3′SuL (carbon (yellow)) represented as sticks. Hydrogen bonds are illustrated as dashes.

Figure 5. Crystal structure of galectin-4N-2′FL complex.

(A) Simulated annealing omit map of galectin-4N binding site with bound 2′FL. Galectin-4N binding site residues (carbon (green)) with the bound 2′FL (carbon (yellow)) depicted in stick representation. The 2|Fo| – |Fc|α_calc map is illustrated in blue mesh at a 1.0 σ contour level and the |Fo| – |Fc|α_calc map is illustrated in red mesh at a 3.0 σ contour level. The omit map was created by the “composite omit map” task of Phenix. (B) Binding site interactions of galectin-4N and 2′FL. Stereo image of galectin-4N binding site depicted in green cartoon representation with binding site residues (carbon (green)) and bound 2′FL (carbon (yellow)) represented as sticks. Possible hydrogen bonds are illustrated as dashes.