Siglec-7 Undergoes a Major Conformational Change When Complexed with the α(2,8)-Disialylganglioside GT1b

Helen Attrill,Akihiro Imamura,Ritu S Sharma,Makoto Kiso,Paul R Crocker,Daan M.F Van Aalten

doi:10.1074/jbc.m601714200

Helen Attrill, Akihiro Imamura + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m601714200

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Abstract

The siglecs are a group of mammalian sialic acid binding receptors expressed predominantly in the immune system. The CD33-related siglecs show complex recognition patterns for sialylated glycans. Siglec-7 shows a preference for alpha(2,8)-disialylated ligands and provides a structural template for studying the key interactions that drive this selectivity. We have co-crystallized Siglec-7 with a synthetic oligosaccharide corresponding to the alpha(2,8)-disialylated ganglioside GT1b. The crystal structure of the complex offers a first glimpse into how this important family of lectins binds the structurally diverse gangliosides. The structure reveals that the C-C' loop, a region implicated in previous studies as driving siglec specificity, undergoes a dramatic conformational shift, allowing it to interact with the underlying neutral glycan core of the ganglioside. The structural data in combination with mutagenesis studies show that binding of the ganglioside is driven by extensive hydrophobic contacts together with key polar interactions and that the binding site structure is complementary to preferred solution conformations of GT1b.

Highlights

Siglecs are a group of evolutionary conserved receptors that constitute the major subgroup of the I-type lectin family [1,2,3]
With the exceptions of myelin-associated glycoprotein (MAG, Siglec-4), found in the nervous system [9, 10], and Siglec-6, expressed on placental trophoblast cells [11], siglec expression appears to be restricted to cells of the hemopoietic and immune systems
Nicoll et al [21], have demonstrated that a b-series ganglioside, GD3,3 when expressed on target cells, was able to mediate interactions with Siglec-7 expressed on NK cells, leading to the suppression of NK mediated cytolytic activity

Summary

EXPERIMENTAL PROCEDURES

Synthesis of GT1b—The GT1b analog (see Fig. 1A) was synthesized from the lactose derivative 2-(trimethylsilyl)ethyl 2,6di-O-benzyl-␤-D-galactopyranosyl-(134)-2,3,6-tri-O-benzylb-D-glucopyranoside using the reaction scheme described by Ishida et al [28]. A gel filtration step was performed using a Superdex 75 26/60 (GE Healthcare) column in 25 mM Tris, pH 8.0, 75 mM NaCl. Crystallization and Data Collection—The protein was concentrated to 8 mg/ml and incubated with a 20-fold molar excess of the GT1b analog. Molecular replacement was performed using AMoRe [31] using the Siglec-7 apo structure PDB code 1O7S. After model refinement and inclusion of waters, the five sugar residues and the trimethylsilyl group of the GT1b analog were built into good qualityFo ϪFc, ␸calc density in the binding site (see Fig. 1B). The final model has an R-factor of 19.5% and an Rfree of 23.4%, with all non-glycine residues in the most favorable and allowed regions of the Ramachandran plot (see Table 1 for statistical analysis). The plasmids were transiently transfected into COS-1 cells using FuGENE

Data collection and refinement statistics

RESULTS

Water molecule

Sugar linkage