Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin

Abstract

Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.

Keywords: DARPin; anthrax toxin; circular dichroism; cytosolic protein delivery; protein stability.

Figure 1

Quantification of Western blots shown in Supplementary Figure S1, measuring the delivery of different LF_N-cargo constructs with PA_wt-sANTXR-Ac2 from a representative experiment. Cargo proteins delivered to the cytosol become biotinylated by cytoplasmic BirA and are subsequently stained with Streptavidin IRDye 680LT. Cytosolic uptake was determined by normalizing signal intensities of cytosolic uptake to the signal of the unspecific interaction of streptavidin with HSP70, which correlates well with the actin signal, and thus can serve as an intrinsic calibration [6]. The delivered cargo DARPin with one internal repeat (labeled “NI₁C”) is used as a control for maximum signal intensity of cytosolic uptake and cells without any delivered cargo (labeled “Cells”) as a negative control. The location of the mutations in the destabilized (dest) DARPins is listed in Table 1, Figure S3b, and [6].

Figure 2

GdnHCl-induced equilibrium unfolding of various cargo DARPins in PBS (pH 7.4) at 20 °C, analyzed by CD spectroscopy. Curves represent a fit to Equation (1). (a) LF_N and the consensus DARPins NI₁C, NI₁C-NI₁C, NI₂C, and NI₂C dest2; (b) NI₃C consensus DARPin and mutated variants of it (NI₃C dest1–4); (c) target-selected LoopDARPins; (d) MBP-binding DARPin off7 and a destabilized off7 variant; (e) JNK-binding DARPins and eGFP-binding DARPin 3G124; (f) NI₃C dest1 variants with repeat-specific mutations: dashed lines represent NI₃C with no mutations (right) and NI₃C dest1 with all mutations (left). Destabilizing mutations (dest1–4) have been described before [6], and their location is listed in Table 1.