Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin

Abstract

Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin (MARTX(Vc)). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6)P(6) or InsP(6)) stimulated the autoprocessing of TcdB dependent upon the CPD (Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314-25321). In this work, the autoprocessing activity of the CPD within MARTX(Vc) is similarly found to be inducible by InsP(6). The CPD is shown to bind InsP(6) (K(d), 0.6 microm), and InsP(6) is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 microm. Processed CPD did not bind InsP(6) indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP(6) binding and 5 lysine and arginine residues resulting in loss of activity at low InsP(6) concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP(6) binding pocket and that the binding of InsP(6) stimulates processing by altering the CPD to an activated conformation. After processing, InsP(6) is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP(6).

FIGURE 1.

Physiological concentrations of InsP₆ stimulate rCPD autoprocessing. Purified rCPD or rCPD C-S (2 μg) were incubated for 1 h at 37 °C in the absence of activator (Control) or in the presence of 100 μm InsP₆ or 1 mm GTPγS. For protease inhibitor assay, rCPD was preincubated for 30 min with 1 mm NEM and then incubated for 1 h in 100 μm InsP₆ or 1 mm GTPγ_S at 37 °C. Proteins were separated by SDS-PAGE (15%) and stained with Coomassie Blue R250. FL indicates full-length unprocessed protein, while P indicates processed protein.

FIGURE 2.

Time course of InsP₆-induced cleavage of rCPD. Purified rCPD was incubated in the presence of 100 μm InsP₆ at 37 °C. Aliquots of 20 μl with 2 μg of rCPD were withdrawn at the indicated time points and mixed with SDS-PAGE loading buffer and boiled to stop the cleavage reaction. Samples were separated by SDS-PAGE (15%) and stained with Coomassie Blue R250. FL indicates full-length unprocessed protein, while P indicates processed protein. rCPD cleavage (%) was semiquantified by densitometric analysis using ImageJ software. The values represent the mean ± S.D. of three independent determinations. A representative gel is shown (A). The obtained data are plotted in the graph (B).

FIGURE 3.

Dependence of autoprocessing on InsP₆ concentration. rCPD (2 μg) was incubated for 2 h at 37°C at the indicated concentrations of InsP₆. Samples were separated by SDS-PAGE and stained with Coomassie Blue R250. FL indicates full-length unprocessed protein, while P indicates processed protein. Autoprocessing was semiquantified by densitometric analysis using ImageJ software. The values represent the mean ± S.D. of three independent determinations. A representative gel is shown (A). The obtained data are plotted in the graph (B).

FIGURE 4.

Dependence of autoprocessing on the rCPD protein concentration. 2-fold dilutions of rCPD as indicated at top were incubated at concentration of InsP₆ indicted below gel for 1 h at 37 °C. Samples were separated by SDS-PAGE and stained with Coomassie Blue R250. FL indicates full-length unprocessed protein, while P indicates processed protein.

FIGURE 5.

InsP₆ binding to rCPD. The ITC binding curve for the interaction of InsP₆ with rCPD variants was measured. The InsP₆ solution was titrated into a cell containing a solution of rCPD variant at constant temperature of 37 °C. The binding curve (lower panels) was obtained from a plot of the heat change from each injection of InsP₆ against the molar ratio of InsP₆ and binding partner (rCPD variant) in the cell (upper panels). In case of cleaved rCPD, rCPD wild-type (15 μm) was preincubated with 2.15μm concentration of InsP₆ for 5 h at 37 °C to obtain the fully processed rCPD protein. The data have been fitted with a nonlinear least squares algorithm, and the dissociation constant (K_d) was calculated.

FIGURE 6.

Residues of CPD important for InsP₆-induced cleavage. A, sequence of CPD domain of MARTX_Vc including cleavage site (arrow) and 8 MARTX_Vc amino acids upstream. Residues mutated are indicated (boxes). The underlined residues are 100% conserved among all 24 CPDs. B, mutant rCPD were incubated in the presence or absence of 0.1 μm InsP₆ as indicated then incubated at 37 °C for 1 h. Autoprocessing was determined by separating samples on SDS-PAGE (15%) and staining with Coomassie Blue R250. FL indicates full-length unprocessed protein, while P indicates processed protein.

FIGURE 7.

Competitive inhibition of rCPD autoprocessing by addition of rCPD mutants. All processing reactions were performed with indicated concentrations of protein and InsP₆ for 2 h at 37°C. In all gels, samples were separated by SDS-PAGE and stained with Coomassie Blue R250 to detect full-length (FL) or processed (P) proteins. A, 2 μg of rCPD (wt) and/or rCPD C-S (or rCPD K3482A) were incubated in the absence or presence of 0.1 μm InsP₆. B, indicated concentrations of rCPD and/or rCPD C-S were incubated with or without indicated concentrations of InsP₆. For both A and B, FL indicates full-length unprocessed protein of both wt rCPD and/or mutant variants, while P indicates processed proteins. C, 2 μg of wt rCPDΔ51 (wtΔ51) and/or rCPD C-S or rCPD K3482A variants were mixed and processing was activated by addition of the indicated concentration of InsP₆. FL indicates the full-length of rCPD C-S or rCPD K3482A, while FLΔ51 marks the full-length size of rCPDΔ51 and PΔ51 marks the processed size of rCPDΔ51. As indicated, the processed form of rCPD C-S or rCPD K3482A would be expected to migrate similar to full-length rCPDΔ51. However, it is clear this processing does not occur as there is no decrease in the amount of FL rCPD C-S or rCPD K3482A.