Glycolipid recognition and binding by Siglec-6 hinges on interactions with the cell membrane

Sialic acid-binding immunoglobulin-type lectins (Siglecs) regulate immune response through interactions with sialylated glycan epitopes on glycoproteins and glycolipids. Human Siglecs count 14 unique proteins allocated to two subsets; group 1 includes four evolutionarily conserved proteins with clear orthologues in all mammals with low sequence similarity and group 2 includes ten rapidly evolving proteins with high sequence similarity and species specificity. In all Siglecs, the recognition and binding of the sialic acid on the glycan target involves a conserved, or canonical, Arg residue. Yet, for a subset of human Siglecs, namely MAG, Siglec-6, and Siglec-11, this Arg appears not to be essential, suggesting that a different binding mechanism may be at play. In this work, we used all-atom molecular dynamics (MD) simulations, binding assays, and mutagenesis to investigate the structural, mechanistic and energetic details of the binding of Siglec-6 to monosialylated gangliosides. Our results show that Siglec-6 relies only partially on its conserved Arg (Arg122) for recognition of membrane-bound gangliosides and that it supplements its binding free energy through interactions with the phospholipids in the membrane surrounding the target epitope. We identified residues Lys126 and Trp127 as key players in this unique membrane interaction, and we confirmed by mutagenesis assays that the loss of these residues abrogates binding. These results provide a step-change in our understanding of the diversification of human Siglecs across group 2, where different members likely evolved as molecular precision tools, to bind specific sialosides by adapting their structure to recognize not only the epitope, but also the biological environment in which it is found.