Monitoring the Kinetics of the pH-Driven Transition of the Anthrax Toxin Prepore to the Pore by Biolayer Interferometry and Surface Plasmon Resonance

Abstract

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

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

“…This single channel unit allows one to follow the kinetics of the anthrax toxin assembly and pore transition reactions (Figs. 7, 8; also see Naik et al 2013). It is worth noting that the acid-induced structural changes accompanying the PA prepore to pore transition alone yield substantial and reproducible signals, allowing one to follow changes in these large observable changes as potential read-outs for inhibitor development (Fig.…”

“…Our laboratories developed a number of novel and entirely unique BLI based platform technologies that will eventually lead to the direct assessment of novel antimicrobial compounds targeting specific molecular transition reactions. The advantages and comparisons of these direct measurement approaches for novel drug discovery are extensively discussed in Naik et al (2013). More importantly, however, useful test compounds that specifically inhibit pH induced transitions will be unique, novel anti-microbial agents because they can potentially be designed to directly target the initial pH dependent molecular transitions that lead to protein toxin entry and in some cases, cell lethality.…”

“…7 below) on BLI biosensor tips (Naik et al 2013). This approach enables one to directly visualize and target the actual molecular transitions that facilitate toxin entry through the PA pore into the cell (Fig.…”

“…Once assembled complexes are released from the small BLI biosensor tips into small volumes (e.g., 2–3 μl), the formation of large complexes (>100 kDa) can be easily verified using electron microscopy (Fig. 9; Naik et al 2013, 2014). …”

Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio‐layer interferometry

“…Samples were then imaged using a JEOL-1200 EXII transmission electron microscope at 100 keV housed at the University of Kansas Medical Campus (KUMC) as described previously. 50 …”