## 2 Glycosphingolipids
It has indeed been noticed that proteins that do not have any sequence similarity (i.e., STxB and CTxB, but also the VP1 capsid proteins of simian virus 40) fold, such as to present binding pockets for the sugars of their GSL receptors in the same position and orientation at the periphery of the pentameric \"doughnut\"-like shapes that they adopt (reviewed in references [6; 23; 24]) (Figure 5). Of note, these binding pockets are located at a distance above the normal plan of the membrane, such that the membrane must bend up at the edges to allow the GSL sugars to reach these sites. It remains to be seen whether this type of molecular organization is also found for the oligomeric gla  
Figure 4: Hypothesis on asymmetric compressive stress leading to negative membrane curvature. Membrane compression of the exoplasmic leaflet to which STxB is bound will force the double-layered plasma membrane to curve in the direction of the toxin. From Reference [20]; courtesy of the Cold Spring Harbor Laboratory Archives. The use of synthetic Gb3 species allowed to show the capacity of STxB to induce tubular membrane invaginations on GUVs only in the presence of single unsaturated acyl chains, and not fully saturated chains [14] (Figure 6). Based on all atom molecular dynamics simulations it was suggested that this was not because STxB lost its capacity to bend membranes as such [50]. Rather, the raft-type membrane patch that was assembled by STxB became too rigid to be bent when it only contained fully saturated Gb3 species [50]. Figure 5: Negative curvature induction based on the geometry of GSL binding sites on lectins. (**a**) All atom molecular dynamics simulation of the STxB homopentamer (gray ribbon structure, side view) on a membrane patch. Gb3 molecules in blue. Note the increment of spontaneous membrane curvature that is observed in silico due to the presence of STxB. (**b**) Representation (extracted from (**a**)) of STxB (side view) with three carbohydrates (blue, green and red). For simplicity, only the three Gb3 binding sites on one monomer are shown. Note that sites 1 (red) and 2 (green) are at the periphery of the protein, while site 3 (blue) faces down under the protein. (**c**) Schematic representation of the situation in (**b**) which illustrates that the membrane (black line) needs to bend up at the edges of STxB to plug the sugars of Gb3 receptor molecules (red and green) into the binding pockets of sites 1 and 2. This would then lead to the generation of negative membrane curvature. (**d**) Overlay of the structures of the following proteins in interaction with GSL receptor analogues: STxB in green with Gb3 (Reference [53]), CTxB in red with GM1 (Reference [54]), the capsid protein VP1 from SV40 in blue (Reference [55]). Note that despite the fact that these proteins do not have any sequence similarity, they fold such that the conserved GSL binding site 2 is presented with the same geometry in space (the sugars of the three structures overlay). It therefore seems likely that this fold was selected by convergent evolution for the same function: to generate negative membrane curvature for building endocytic sites for clathrin-independent endocytosis. Reproduced with permission from Toxins, Ref. [24]. Of note, for the highly related cholera toxin, endocytic and retrograde trafficking into cells was much more efficient with single unsaturated GM1 acyl chain species, when compared to the fully saturated ones [56]. This finding indicated that the capacity to organize a bending-compatible raft-type nanodomain was indeed critical for the biological effect of the toxins.