Membrane invagination induced by Shiga toxin B-subunit: from molecular structure to tube formation

Abstract

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.

Mechanism of Shiga Toxin Clustering on Membranes

“…When this phase separation occurs on membranes with low values of membrane tension, the small increment of curvature induced by each toxin particle, which has been predicted from atomistic Molecular Dynamics simulations 14 to be 0.035 nm –1 , creates a spontaneous curvature in the toxin-rich domains 40 that subsequently drives their tubular invagination, 41,42 as seen in Figure 1B.…”

“…(14) The question thus arises whether such a curvature imprint can generate an attractive force. Indeed, for highly curved particles, an attractive curvature-induced force has been described previously.…”

“…12,15 STxB clearly falls into this category with a low contact angle around 7°, resulting from a spontaneous curvature radius that is 5-fold bigger than the protein’s size. 14 We can therefore exclude induced membrane curvature as the driving force for STxB clustering.…”

“…Nor was the hydrophobic core of the membrane affected by bound STxB. Furthermore, using atomistic molecular dynamics simulations, we recently obtained evidence that suggests that membrane-bound STxB creates a small increment of curvature, 14 which would be expected to yield a repulsive force between toxin molecules. 12,15 However, our simulations and experiments show that reducing the rigidity of the bound STxB nanoparticles or displacing them from the membrane surface eliminates the clustering process.…”

“…Description of the thermal Casimir force between membrane inclusions as presented in the literature and our calculations using the proximity force approximation of the thermal Casimir force between a pair of polygonal particles at separations that are small compared to their size, the dissipative particle dynamics simulations of nanoparticles adsorbed to a fluctuating membrane, showing that only when the nanoparticles suppress the membrane fluctuations do they experience a clustering force, reanalysis of trajectories from our previously published molecular dynamics (MD) simulations 14,18 showing that the binding of STxB to the membrane does not change the lipids’ chain order parameter nor their rotational diffusion, experimental protocol for adding the STxB toxin particles to the vesicles and cells, estimation of the membrane coverage due to STxB binding to Gb3 lipids, FCS experiments that are used to measure the decrease in diffusion as the toxin nanoparticles aggregate, and complete organic synthesis reactions for the PEG-modified Gb3 lipids (PDF)…”