Homo sapiens Galectin-1

Arthur Lian '19 and Weichen Zhao '20

Contents:

I. Introduction

Galectins are a phylogenetically conserved family of lectins, carbohydrate-binding proteins, that are characterized by their affinity for B-galactosidases (Camby et al., 2006). Differentially expressed by various normal and pathological tissues, Homo sapien galectin-1 (hGal-1) has been associated with a wide range of biological activities. The most studied hGal-1 functions include cell adhesion, cell growth, apoptosis, immunological responses, RNA processing, tumor progression, and nerve regeneration.

Carbohydrates are not the only ligand hGal-1 is capable of binding; hGal-1 has also been observed to interact extensively with proteins. Different binding activities of hGal-1 are separated by cell membrane - the lectin activity is mostly observed to be extracellular whereas the protein-protein interaction is intracellular. The most prominent intracellular protein-protein interaction hGal-1 is capable of is with the oncogenic H-Ras protein, whose mutated form has shown to be implicated in tumor formation (Lopez-Lucendo et al., 2004). Considering hGal-1’s versatility with ligand binding and involvement in pathological pathways, designing ligands to block the activity of hGal-1 can potentially block the malignant biological pathways that hGal-1 is involved in. Therefore, understanding the structure of hGal-1 is a major component of rational drug design. With this tutorial, we are showing the basic structure of hGal-1 and its carbohydrate-binding activities. We will also show two mutations of hGal-1 (C2S and R111H) that weakens its carbohydrate-binding abilities.

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(Camby et al., 2006)

II. General Structure

hGal-1 is a with 135 amino acids and a carbohydrate-binding domain (CBD) in each monomer subunit (Lopez-Lucendo et al., 2004; Barondes et al., 1994). hGal-1 adopts a "jelly-roll" topology that involves a sandwich consisting of two antiparallel sheets of five (F1-F5) and six (S1-S6) over a hydrophobic core. The N and C termini of each monomer are positioned at the dimer interface and the are located at the far ends of the same face.

The integrity of the dimer is maintained by both the hydrophobic core at the monomer interface and that within each monomer. The hydrophobic core at the interface is maintained by four hydrogen bonds between and from both S1 strands in the anterior face, and five hydrogen bonds involving residues , ,and from the F1 strands in the opposing face. In addition, the internal hydrophobic core is formed by nonpolar residues from both the S1 ( , and ) and F1 ( , , ).

III. Carbohydrate Binding

The CBD encompasses B-strands S4-S6a/b on the concave face of one B-sheet. The amino acid residues involved in interactions with the bound disaccharide are Arg73 , His44 ,Arg48 Val59 , Asn46 , Asn61 , Trp68 ,and Glu71 (Lopez-Lucendo et al., 2004).

The majority of the interactions between hGal-1 and carbohydrates are formed between the side-chains of certain amino acid residues and the oxygen atoms in sugars, specifically with His44, Arg48, and Asn46. Asn46 is also implicated in a water-mediated hydrogen bond with the hydroxyl group of . The ring oxygen O5 participates in hydrogen bonding with and . The planar side chains of residues Trp68 and His52 are instrumental in the correct alignment of the sugar to the binding site. Prominently, pi stacking interactions between Trp68 and carbons of the B face of the galactose ring are crucial in distinguishing galactose from glucose. Carbohydrate-protein interactions are structurally supported by ionic protein-protein interactions between residues Arg48 , Asp54 ,Arg73 , Glu71 which form three salt bridges that position the protein side-chains in the optimal orientation for sugar binding.

IV. Mutations

One of the hGal-1 mutants that may be clinically significant is the C2S mutant. Because the oxygen from the hydroxyl group in Ser is a weaker reducing agent than the sulfur from the thiol group in Cys, the mutant is in a less reduced state and thus should be more resistant to oxidation. The C2S mutant has been observed to be more stable than wild type hGal-1 (WT) (Hirabayashi et al., 1991) and such stability is promising in terms of clinical applications.

A significant conformational change between the WT and the C2S mutant involves the orientation of Asp123 and the connections it makes with Val131 and Cys2 (Or Ser2, in the case of C2S). The directs Asp123 towards F1 and forms a hydrogen bond with Val131. In the , Asp123 faces toward Ser2 and distorts the connecting loops. Though this conformational change is distant from the CRD, it significantly lowers hGal-1’s affinity for carbohydrates.

Likewise, such conformational change is also present in the . Furthermore, the R111H substitution is also responsible for a direct alteration of the CRD. In Arg111 forms hydrogen bonds with the hydroxyl group of Ser62 and with the carbonyl group of Thr70. In contrast, with the , Ser62 and Thr70 establish hydrogen bonds with the ring of His111 and consequently distorts the loops connecting F4-F5, F3-F4, F3-F6b, S5-S6a, and S4-S5. The distortion shifts - two highly conserved amino acid residues involved in carbohydrate recognition - and weakens its ability to bind carbohydrates.

VI. References

Camby, I., Mercier, M. L., Lefranc, F., and Kiss, R. (2006). Galectin-1: a small protein with major functions. Glycobiology, 16(11), pp. 137-157.

Hirabayashi, J. & Kasai, K.-I. (1991). Effect of amino acid substitution by site-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa b-galactoside-binding lectin. Jounal of Biological Chemistry, 266, pp. 23648–23653.

Lopez-Lucendo, M. F., Solis, D., Andre, S., Hirabayashi, S. A. J., Kasai, K., Gabius, H., and Romero, A. (2004). Growth-regulatory Human Galectin-1: Crystallographic Characterisation of the Structural Changes Induced by Single-site Mutations and their Impact on the Thermodynamics of Ligand Binding. Journal of Molecular Biology, 343(4), pp. 957-970.

Barondes, S. H., Cooper, D., Gitt, M. A., and Leffler, H. (1994). Galectins: Structure and Function of a Large Family of Animal Lectins. The Journal of Biological Chemistry, 269(33), > 20807-20810.

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