Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs

Abstract

Bacterial toxin or viral entry into the cell often requires cell surface binding and endocytosis. The endosomal acidification induces a limited unfolding/refolding and membrane insertion reaction of the soluble toxins or viral proteins into their translocation competent or membrane inserted states. At the molecular level, the specific orientation and immobilization of the pre-transitioned toxin on the cell surface is often an important prerequisite prior to cell entry. We propose that structures of some toxin membrane insertion complexes may be observed through procedures where one rationally immobilizes the soluble toxin so that potential unfolding ↔ refolding transitions that occur prior to membrane insertion orientate away from the immobilization surface in the presence of lipid micelle pre-nanodisc structures. As a specific example, the immobilized prepore form of the anthrax toxin pore translocon or protective antigen can be transitioned, inserted into a model lipid membrane (nanodiscs), and released from the immobilized support in its membrane solubilized form. This particular strategy, although unconventional, is a useful procedure for generating pure membrane-inserted toxins in nanodiscs for electron microscopy structural analysis. In addition, generating a similar immobilized platform on label-free biosensor surfaces allows one to observe the kinetics of these acid-induced membrane insertion transitions. These platforms can facilitate the rational design of inhibitors that specifically target the toxin membrane insertion transitions that occur during endosomal acidification. This approach may lead to a new class of direct anti-toxin inhibitors.

Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domain

“…We previously published a methodology to assemble LF N -PA pore complexes while avoiding aggregation by immobilizing PA pores before solubilizing the hydrophobic tip with lipid bilayer nanodiscs [ 22 , 23 , 24 , 25 ]. After immobilization, the PA prepores were transitioned into pores using a urea/37°C pulse methodology, exposing the aggregation-prone pore tip.…”

“…After immobilization, the PA prepores were transitioned into pores using a urea/37°C pulse methodology, exposing the aggregation-prone pore tip. The nanodisc formed around the hydrophobic pore tip while the complex was immobilized [ 22 , 23 , 25 , 26 ]. A schematic of this methodology is shown in Figure 1 A.…”

“…One of the main thrusts of this work has been to demonstrate that we can routinely obtain highly-pure engagement complexes (multiply- or singly-bound LF N ) using an immobilization bead-based protocol and nanodisc technology without using columns to purify the final complexes [ 23 , 25 ] and minimizing detergent influences on structure [ 31 , 32 ]. Even at 17 Å resolution, the variability of the domain 4 densities for the LF N -PA pore indicates this region is intrinsically flexible [ 17 ], ruling out the possibility that this flexibility is due to grid adherence constraints.…”

“…Heterogeneous LF N -PA-nanodisc complexes were formed and purified as previously described [ 22 , 25 ]. In brief, E126C LF N was immobilized by coupling E126C LF N to activated thiol sepharose 4B beads (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) in Assembly Buffer (50 mM Tris, 50 mM NaCl, pH 7.5) at 4 °C for 12 h. One hundred microliters (100 μL) of 0.2 μM heptameric WT PA prepore was then added to 50 μL of LF N bead slurry.…”

Membrane Protein Folding & Lipid Interactions: Theory & Experiment

“…Bacterial toxins— another traditional subject for exploring lipid–protein interaction—are represented by studies of pore assembly of perfringolysin O [13], thermodynamics of the insertion and refolding of diphtheria toxin translocation domain [14], and lipid nanodisc-based methodology to study inserted bacterial toxins [15]. Structural motifs involved in membrane pore formation by bacterial toxins are reviewed and compared to those involved in mitochondrial outer membrane permeabilization by Bcl-2 proteins during apoptosis [16].…”