The adenylate cyclase toxin RTX domain follows a series templated folding mechanism with implications for toxin activity

Abstract

Folding of the Repeats-in-toxin (RTX) domain of the bacterial adenylate cyclase toxin-hemolysin (CyaA) is critical to its toxin activities and the virulence of the whooping cough agent Bordetella pertussis. The RTX domain (RD) contains five RTX blocks (RTX-i to RTX-v) and their folding is driven by the binding of calcium. However, the detailed molecular mechanism via which the folding signal transmits within the five RTX blocks remains unknown. By combining single molecule optical tweezers, protein engineering, and toxin activity assays, here we demonstrate that the folding of the RD follows a strict hierarchy, with the folding starting from its C-terminal block RTX-v and proceeding towards the N-terminal RTX-i block sequentially. Our results reveal a strict series, templated folding mechanism, where the folding signal is transmitted along the RD in a series fashion from its C terminus continuously to the N terminus. Due to the series nature of this folding signal transmission pathway, the folding of RD can be disrupted at any given RTX block, rendering the RTX blocks located N-terminally to the disruption site and the acylation region of CyaA unfolded and abolishing CyaA's toxin activities. Our results reveal key mechanistic insights into the secretion and folding process of CyaA and may open up new potential avenues towards designing new therapeutics to abolish toxin activity of CyaA and combat B. pertussis.

Keywords: adenylate cyclase; bacterial toxin; optical tweezers; protein folding; single-molecule biophysics.

Figure 1

Probing the unfolding and folding of RD using optical tweezer.A, scheme of the domain structure of CyaA. CyaA is a large multidomain protein that harbors different functional domains. RD is located at the C-terminal of CyaA and is comprised of five RTX blocks. Each block consists of RTX nonapeptide tandem repeat sequences followed by non-RTX linker sequence. The CR3-binding site binds CR3 integrin via two binding interfaces, which are between RTX-i and ii as well as between RTX-ii and iii. B, 3D structure of the RTX-i-v derived from the cryo-EM structure of RTX751 (PDB ID 7USL) (26). Ca²⁺ ions are shown as gray spheres. C, scheme of the OT experiment on NuG2-RTX-i-v-NuG2. NuG2-RTX-iii-v-NuG2 is coupled to two DNA handles (functionalized with biotin and digoxigenin, respectively) through thiol-maleimide reaction. The DNA-protein chimera is then captured by two polystyrene beads coated with streptavidin and anti-digoxigenin via specific ligand–receptor interactions. One bead is held by the glass micropipette and the other is trapped by the laser trap. By moving the laser trap, the investigated protein can be stretched and relaxed to induce unfolding and folding. D, representative F-D curves showing the unfolding-folding events of RTX-i-v. Individual (un)folding events are colored. Dotted lines are pseudo-WLC fits. When the second unfolding event (in blue) occurs at relatively lower forces, three additional unfolding events can be clearly resolved (curve 1 and inset). However, when the second unfolding event occurs at relatively higher forces, fewer unfolding events are resolved (curve 2). It is of note that the unfolding of the fingerprint domain NuG2 typically occurs at higher forces (Fig. S1). We often observed that RTX-i-v unfolds prior to NuG2, so that we can limit the stretching so that the unfolding of NuG2 does not occur, and F-D curves only display the unfolding-folding events of RTX-i-v. CR3, complement receptor 3; RD, RTX domain; RTX, Repeats-in-toxin.

Figure 2

Mechanical folding/unfolding of RTX-iii-v.A, representative F-D curves of RTX-iii-v in the presence of 10 mM Ca²⁺ at a pulling speed of 50 nm/s. Three distinct unfolding and refolding events are observed. The red unfolding events show a clear two-state unfolding behavior. Unfolding events of RTX-v can be readily identified by its characteristic ΔLc of 45 nm, while unfolding events of RTX-iii and iv show similar ΔLc. The unfolding events of RTX-iv can be identified by its characteristic multistate unfolding. The (un)folding events of RTX-iii, iv, and v are colored in red, cyan, and green, respectively. B, force-extension relationships of the (un)folding of RTX-iii. WLC fits to the data shows a ΔLc of 39.5 ± 0.6 nm. C, histograms of unfolding and refolding forces of RTX-iii at a pulling speed of 50 nm/s. The unfolding and refolding force is 14.1 ± 1.4 pN (average ± SD, n = 383) and 3.5 ± 1.0 pN, respectively. D, consecutive stretching-relaxation cycles allows for assigning the folding events to individual RTX blocks. F-D, force-distance; RTX, Repeats-in-toxin.

Figure 3

Mechanical unfolding/folding signatures of RTX-ii.A, representative F-D curves of RTX-ii-v. Individual (un)folding events are colored. B, force-extension relationship of the (un)folding of RTX-ii. WLC model fitting yields a ΔLc of 41.6 ± 0.6 nm. C, histograms of unfolding and folding forces of RTX-ii at a pulling speed of 50 nm/s. The average unfolding force and refolding force are 26.3 ± 4.3 pN and 3.4 ± 1.0 pN, respectively. D, repeated stretching-relaxation curve of RTX-ii-v allows for discerning the unfolding and folding order of the RTX blocks. F-D, force-distance; RTX, Repeats-in-toxin.

Figure 4

RTX-i is the first RTX block to unfold but the last to refold.A, F-D curves showing the unfolding-refolding event of RTX-i. RTX-i unfolds and refolds via an intermediate state. Dotted lines are pseudo WLC fits that help identify the intermediate. B, unfolding and refolding force histogram of RTX-i. C, repeated stretching-relaxation curves of RTX-i-v shows that the unfolding of RTX-i-v occurs in the order of RTX-i to RTX-v, while the refolding occurs in the reverse order from RTX-v to i. F-D, force-distance; RTX, Repeats-in-toxin.

Figure 5

The transmission of the folding signal can be blocked by the RS insertion. A-D, the schematic of the domain structure (left) and the representative F-D curves (right) of different RS insertion RTX variants (A: RTX-iv-v-RS1528; B: RTX-iii-v-RS1528; C: RTX-i-v-RS1261; D: RTX-i-v-RS1137). In the schematics, RTX blocks are colored in cyan, and RS insertions are colored in blue. In F-D curves, unfolding and folding events are colored according to their identities, that is, RTX-v in green, RTX-iv in cyan, RTX-iii in red, and RTX-ii in blue. Zoomed view of the folding events of RTX blocks are shown in insets. Folding events of the fingerprint domain NuG2 are indicated by circles, while unfolding events of NuG2 occurred at higher forces (>30 pN). F-D, force-distance; RTX, Repeats-in-toxin.

Figure 6

Structural and functional properties of the RS-insertion CyaA mutants.A and B, far UV CD spectra of CyaA and its RS-insertion mutants in the absence of Ca²⁺(A) and in the presence of 2 mM Ca²⁺ ions (B). C, binding and cytotoxic activities of CyaA and its RS-insertion variants on THP-1 cells. The binding was determined in D-MEM (1.9 mM Ca²⁺) as the amount of cell-associated AC enzyme activity after incubation of 1 × 10⁶ cells with 1 μg/ml of the indicated CyaA proteins for 30 min at 4 °C. AC domain delivery of CyaA was assessed by determining the intracellular cAMP concentration generated at 37 °C in THP-1 cells (1.5 × 10⁵) after 30 min of incubation with four different CyaA concentrations from the linear range of the dose-response curve (250, 125, 62.5, and 31.25 ng/ml). The activities of CyaA at each of the toxin concentrations was taken as 100% for calculation of % of cAMP generated by CyaA-RS proteins used at each of the four indicated toxin concentrations. The % values were then averaged for each CyaA RS variant and the means are given. D, the activities of CyaA and its RS-insertion variants on red blood cells. Sheep erythrocytes (5 × 10⁸/ml) were incubated at 37 °C in the presence of 2 mM Ca²⁺ with 1 μg/ml of the purified CyaA proteins. After 30 min, aliquots were taken to determine the cell-associated AC activity (binding) and the AC activity internalized into erythrocytes and protected from digestion by externally added trypsin (Invasive AC). For determination of hemolytic activity, sheep erythrocytes (5 × 10⁸/ml) in TNC buffer were incubated at 37 °C with 10 μg/ml of CyaA proteins for 3 h as the amount of released hemoglobin was measured as A₅₄₁. Activities are expressed as percentages of intact CyaA activity and represent averages ± SDs. N = 4. Two independent toxin preparations were analyzed in all assays. AC, adenylate cyclase; DMEM, Dulbecco’s modified Eagle’s medium.