The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications.

The bacterial Shiga toxin is composed of an enzymatically active A-subunit, and a receptor-binding homopentameric B-subunit (STxB) that mediates intracellular toxin trafficking. Upon STxB-mediated binding to the glycolipid globotriaosylceramide (Gb₃) at the plasma membrane of target cells, Shiga toxin is internalized by clathrin-dependent and independent endocytosis. The formation of tubular membrane invaginations is an essential step in the clathrin-independent STxB uptake process. However, the mechanism by which STxB induces these invaginations has remained unclear. Using a combination of all-atom molecular dynamics and Monte Carlo simulations we show that the molecular architecture of STxB enables the following sequence of events: the Gb₃ binding sites on STxB are arranged such that tight avidity-based binding results in a small increment of local curvature. Membrane-mediated clustering of several toxin molecules then creates a tubular membrane invagination that drives toxin entry into the cell. This mechanism requires: (1) a precise molecular architecture of the STxB binding sites; (2) a fluid bilayer in order for the tubular invagination to form. Although, STxB binding to the membrane requires specific interactions with Gb₃ lipids, our study points to a generic molecular design principle for clathrin-independent endocytosis of nanoparticles.

Model biomolecular condensates have heterogeneous material properties that are tuned by the number and distribution of their constituent proteins’ sticky binding sites.

Globotriaosylceramide (Gb₃) is a glycosphingolipid present in the plasma membrane that is the natural receptor of the bacterial Shiga toxin. The unsaturation level of Gb₃ acyl chains has a drastic impact on lipid bilayer properties and phase behaviour, and on many Gb₃-related cellular processes. For example: the Shiga toxin B subunit forms tubular invaginations in the presence of Gb₃ with an unsaturated acyl chain (U-Gb₃), while in the presence of Gb₃ with a saturated acyl chain (S-Gb₃) such invagination does not occur. We have used all-atom molecular dynamics simulations to investigate the effects of the Gb₃ concentration and its acyl chain saturation on the phase behaviour of a mixed bilayer of dioleoylphosphatidylcholine and Gb₃. The simulation results show that: (1) the Gb₃ acyl chains (longer tails) from one leaflet interdigitate into the opposing leaflet and lead to significant bilayer rigidification and immobilisation of the lipid tails. S-Gb₃ can form a highly ordered, relatively immobile phase which is resistant to bending while these changes for U-Gb₃ are not significant. (2) At low concentrations of Gb₃, U-Gb₃ and S-Gb₃ have a similar impact on the bilayer reminiscent of the effect of sphingomyelin lipids and (3) At higher Gb₃ concentrations, U-Gb₃ mixes better with dioleoylphosphatidylcholine than S-Gb₃. Our simulations also provide the first molecular level structural model of Gb₃ in membranes.