Multiscale Molecular Dynamics Studies Reveal Different Modes of Receptor Clustering by Gb3-Binding Lectins

Abstract

The recognition of carbohydrate receptors on host cell membranes by pathogenic lectins is a crucial step in the microbial invasion. Two bacterial lectins, the B-subunit of Shiga toxin from Shigella dysenteria (StxB) and lectin I from Pseudomonas aeruginosa (LecA), are specific to the same galactolipid–globotriaosylceramide (Gb3). In this study we present a coarse-grained (cg) model of Gb3, which we further apply to unravel the molecular details of glycolipid binding by two lectins on the surface of a DOPC/cholesterol/Gb3 bilayer. In cg molecular dynamics simulations with time scales of dozens of microseconds, Gb3 was randomly distributed. The binding of both StxB or LecA is accompanied by Gb3 clustering in a cholesterol environment and with exclusion of DOPC in protein vicinity. StxB being bound by all 15 binding sites induced membrane bending, while LecA interacted with two out of four binding sites for most of the time causing a smaller inward curvature of the model membrane. Stable interactions occurred preferably when LecA was normal to the membrane surface. Furthermore, all-atom simulations revealed that LecA bound Gb3’s headgroup at only one out of two possible conformations of the carbohydrate moiety observed at protein-free conditions. The results shed light on the mechanism of interactions between two lectins and Gb3 on the membrane surface and offer a coarse-grained model to study more complex systems at large spatiotemporal scales.

Structural Diversities of Lectins Binding to the Glycosphingolipid Gb3

“…Furthermore, the firm binding observed when Gb3 is inserted in the membrane, such as on Burkitt Lymphoma cells, was rationalized since the Gb3 glycosphingolipid geometry promotes the parallel presentation of neighbouring trisaccharide heads, fully compatible with multivalent binding by LecA. Further modelling of the binding of LecA with Gb3 embedded in the extracellular leaflet of a lipid bilayer confirmed the likely clustering of αGal14βGal by this lectin ( Kociurzynski et al, 2021 ).…”

“…LecA has been shown to play a crucial role in the internalization of the whole bacterium into host cells. LecA binding and clustering of Gb3 induces negative membrane curvature ( Kociurzynski et al, 2021 ) resulting in the membrane engulfment of the bacterium via the “lipid zipper” mechanism ( Eierhoff et al, 2014 ). Experiments on several epithelial cell lines confirmed the dependence of this internalization on the presence of both LecA at the bacterial outer membrane and Gb3 in the host cell membrane.…”

“…It does not recognize lactose and other βGal-containing oligosaccharides since His50 would create a steric conflict with the second residue, but the presence of an hydrophobic pocket on the protein surface close to the anomeric position of galactose provides higher affinity for β-galactose functionalized with aromatic ring ( Rodrigue et al, 2013 ). LecA has four binding sites, and due to its topology, two are associated side by side in perfect orientation for binding to Gb3 in membrane ( Kociurzynski et al, 2021 ). This results in avidity that is much stronger than local affinity, with values in the nM range when evaluated with multivalent ligands ( Bernardi et al, 2013 ).…”

Computational Approaches to Explore Bacterial Toxin Entry into the Host Cell

“…The forcefield of these methods is continually being optimized to make better predictions of realistic systems [ 52 ]. The Martini model, a popular CG model that maintains chemical specificity [ 53 ] ( Figure 1 B), has been used to investigate the effect of CTxB and its cellular receptor, i.e., GM1 on lipid bilayer phases and structures [ 54 , 55 ], and the binding of STxB and lectin I from Pseudomonas aeruginosa to membranes containing Gb3 [ 56 ]. Flores-Canales et al [ 57 ] have created a CG model based on atomistic structure, to study the interactions of the diphtheria toxin translocation domain with biomembranes.…”

Therapeutic Uses of Bacterial Subunit Toxins

“…The microvascular endothelial cell raft and non-raft Gb 3 fatty acid composition has recently been found to vary, with saturated species restricted to the raft and unsaturated species to the non-raft fraction [26]. Moreover, different Gb 3 fatty acid binding preferences of distinct Gb 3 binding ligands can result in differential raft membrane Gb 3 clustering [27].…”