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Small Molecule-Induced Allosteric Activation of the Vibrio cholerae RTX Cysteine Protease Domain

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Science  10 Oct 2008:
Vol. 322, Issue 5899, pp. 265-268
DOI: 10.1126/science.1162403

Abstract

Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP6), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP6. InsP6 binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP6 binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

Most secreted bacterial toxins are produced as inactive precursors that become proteolytically activated upon entering a eukaryotic cell (1). A select group of these toxins undergo autoproteolysis upon entry into the host cytosol, resulting in the release of their effector domains (2, 3). The Vibrio cholerae RTX (repeats in toxin) is a member of the multifunctional autoprocessing RTX (MARTX) family of toxins, which all contain a cysteine protease domain (CPD) predicted to mediate the autoproteolytic activation of the secreted protoxin upon entry into the eukaryotic cytosol (4). Almost all clinical and environmental isolates of V. cholerae produce RTX (5), which enhances virulence and colonization in murine models of V. cholerae infection (6, 7). RTX autoprocessing is thought to release its actin-disrupting effector domains from the target cell's plasma membrane into the cytosol (4) (fig. S1). Although autoproteolysis is essential for RTX toxin function (3), the mechanism of RTX CPD activation is unclear.

It has recently been shown that the small molecules guanosine 5′-triphosphate (GTP) and inositol hexakisphosphate (InsP6) stimulate autoprocessing of the RTX CPD (3, 8); also, InsP6 activates a distantly related protease domain in toxin B of Clostridium difficile (2, 9). We tested the ability of guanosine 5′-O-(3′-thiotriphosphate) (GTP-γ-S), InsP6, and InsP6 metabolites to activate V. cholerae RTX CPD autocleavage in vitro. InsP6 potently activated autocatalysis, with a half-maximal autocleavage concentration (AC50) of 0.9 nM versus 0.19 μM, 0.72 μM, and 240 μM for InsP(1,4,5,6)4, InsP(1,3,4,5,6)5, and GTP-γ-S, respectively (Fig. 1A and fig. S3). We confirmed the interaction between InsP6 and RTX CPD using surface plasmon resonance (SPR), which indicated that InsP6 binds to the RTX CPD with an equilibrium binding affinity constant (Kd) of 1.3 ± 0.2 μM (Fig. 1B).

Fig. 1.

InsP6 activates the V. cholerae RTX CPD and the architecture of the InsP6-CPD complex. (A) Activation of RTX cysteine protease domain autocleavage by InsP6. Recombinant RTX CPD (amino acids 3391 to 3650) was incubated with the indicated concentrations of InsP6 for 2 hours, and autocleavage was assessed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining (representative gel inset). Reactions were performed in triplicate, and the amount of autocleaved protein relative to the total protein amount was analyzed by densitometry, averaged and plotted versus concentration of InsP6. 50% of the wild-type CPD was autoprocessed (AC50) at 0.85 ± 0.02 nM InsP6 (mean ± SD). (B) SPR analysis of InsP6 binding to wild-type biotinylated CPD immobilized on a streptavidin-coupled surface. Representative sensorgram (inset) shows the binding of InsP6 to the CPD-bound surface over a concentration of 0.1 to 100 μM. Equilibrium binding analysis indicates a Kd of 1.3 ± 0.2 μM(SD). (C) Structure of the CPD-InsP6 complex viewed from above the InsP6-binding site. (D) A view of the structure rotated ∼135° to show the active site. The InsP6-binding and active sites are separated by a 22–amino acid β-hairpin structure (β-flap). InsP6 and the side chains of the catalytic dyad (Cys140/His91) are shown as stick models.

To gain structural insight into the InsP6-mediated activation of RTX CPD, we purified an autocleaved minimal RTX CPD catalytic domain for cocrystallization with InsP6 (10). We determined the structure of the RTX CPD at a resolution of 2.1 Å, consisting of amino acids 5 through 203 (with residue 1 being the P1′ alanine and P1′ referring to the residue C-terminal to the scissile bond). The protease domain comprises a seven-stranded beta sheet, with five central parallel strands and two antiparallel capping strands (Fig. 1, C and D). Two helices flank one side of the sheet, lying diagonally in a groove created by a ∼90° twist of the sheet, and a third helix caps the other side of the sheet. The Cys-His catalytic dyad (C140 and H91) lies at the C-terminal ends of the central D and E β strands. The overall structure is reminiscent of the clan CD family of cysteine proteases, which suggests a common ancestor for this family of enzymes. Comparison with the two most closely related known protease structures, human caspase-7 (11) and Porphyromonas gingivalis gingipain-R (12) (fig. S4), reveals that two C-terminal helices have been replaced by a three-strand flap. This “β-flap” structure forms a cleft in which we identified electron density for a single InsP6 molecule (fig. S5).

The InsP6-binding pocket is lined with basic residues, burying approximately 890 Å2 of surface area (Fig. 2A). Nine of the twelve residues that directly interact with InsP6 via hydrogen bond contacts are positively charged, with a core of six residues (K54, R85, S136, R171, K183, and K200) forming the bottom of the pocket and five others surrounding the InsP6 molecule (T28, R29, H55, H134, S169, and R182) (Fig. 2B and fig. S6) (13). K195 covers the top of the InsP6 molecule, interacting with the C1, C5, and C6 phosphate groups. The protein–InsP6 interface is further stabilized by a network of water molecules between InsP6 and the β-flap. On the opposite side of the β-flap is the catalytic dyad and a large hydrophobic pocket for the P1 amino acid (the residue N-terminal to the scissile bond) (Fig. 2C). The β-flap contributes three of the twelve hydrophobic amino acids that line one side of the P1 pocket, and helix 1 and two central beta sheets (D and E) contribute the remainder (Fig. 2D). The surface properties and size of the P1 substrate pocket are consistent with the cleavage of RTX CPD after an N-terminal leucine (3). The final N-terminal residue observed in our structure is I5 (the P5′ position) (Fig. 2C), which lies ∼14 Å from the catalytic cysteine. No electron density was observed for the P1′ through P4′ positions, suggesting that they do not make strong contacts with the protease and may minimally contribute to substrate specificity.

Fig. 2.

The InsP6-binding and active sites. (A) Electrostatic surface potential of the CPD as viewed from above the InsP6-binding site. Blue denotes a positively charged surface; red denotes a negatively charged surface. InsP6 is shown in the binding site as a stick model. (B) Close-up view of the InsP6-binding site. Side chains that directly interact with InsP6 are labeled and shown as yellow sticks. The electron density for InsP6 (2FobsFcalc) is contoured at 2σ. (C) Surface topology of the CPD active site. The P1 substrate pocket, C140, and H91 are highlighted in orange, yellow, and blue, respectively. The N terminus is shown as a yellow ribbon, terminating at Ile5 and highlighting the threading of this region along the surface of the core domain. The remaining residues not visible at the N terminus are depicted as a yellow dashed line to illustrate the approximate positioning of the chain during catalysis. (D) Close-up view of the P1 substrate pocket. Amino acids that line the pocket are labeled and colored orange. InsP6 is shown as in (B) to demonstrate the position of the catalytic site with respect to the InsP6-binding site.

The InsP6-binding site is structurally segregated from the active site, which clearly indicates that InsP6 does not act as a cofactor for catalysis. Indeed, kinetic analyses revealed that InsP6 binding is independent of substrate binding to the active site and that the concentration of InsP6 does not alter the affinity of the InsP6-bound enzyme for its substrate (fig. S7). Because RTX CPD activity was strictly dependent on InsP6 in these analyses, we hypothesized that InsP6 binding may regulate exposure of the active site. We therefore tested the ability of a fluorescent maleimide derivative to alkylate the catalytic cysteine of wild-type CPD and a mutant CPD lacking two InsP6-interacting residues (R182Q/K183N) (14). Although weak fluorescent labeling was observed for both wild-type and mutant CPD in the absence of InsP6, dose-dependent labeling of the active-site cysteine was observed in the presence of InsP6 only for the wild-type CPD (fig. S8). Thus, productive binding of InsP6 is required to expose the active-site cysteine of the protease to substrates and inhibitors. Consistent with this observation, pretreatment of wild-type CPD immobilized on an SPR chip with N-ethylmaleimide (NEM) alone failed to block InsP6-induced autocleavage of the CPD from the chip (fig. S9). Simultaneous treatment of the CPD with InsP6 and NEM, however, inhibited InsP6-induced autocleavage, which indicated that NEM can only react with the active-site cysteine in the presence of InsP6. These results strongly suggest an allosteric mechanism of activation in which the active site is disordered or occluded in the absence of InsP6, a mode of regulation that likely protects the protease active-site sulfhydryl until the toxin enters a eukaryotic cell.

Inspection of the structure suggested that the β-flap, which lines the side of the InsP6-binding cleft closest to the catalytic site, may contribute to enzyme activation by properly ordering the P1 pocket and active site. Among the many side chains that coordinate InsP6, the β-flap contains three residues (R182, K183, and K195) that form a three-pronged “clamp” above and below InsP6 (Figs. 2A and 3A). We tested the effect of the mutations R182Q, K183N, and K195N on both InsP6 binding (fig. S10) and catalytic activity (fig. S11) relative to wild-type CPD (Fig. 3A). Mutation of K183 and K195 abrogated both InsP6 binding and autocleavage activity (Fig. 3A, left table), whereas mutation of R182 only moderately reduced InsP6 binding but dramatically decreased autocatalysis as compared with the wild type. R182 not only binds InsP6, it also engages in structurally stabilizing hydrogen-bonding interactions with D24 (fig. S12). This may explain the more considerable impact of the R182Q mutation on catalysis rather than InsP6 binding, because R182 may primarily fine-tune the structure of the β-flap and may contribute nominally to binding InsP6. Thus, diminution of InsP6 binding on one side of the flap clearly reduces catalysis mediated by residues on the opposite side of the flap.

Fig. 3.

β-Flap mutations decouple CPD autocatalysis and RTX activity from InsP6 binding. (A) Comparison of autocleavage efficiency (AC50) versus InsP6 binding (Kd) measured by SPR for mutations in the InsP6-binding site (left table) and β-flap (right tables, top and bottom). The β-flap region of the CPD is rainbow-colored, starting with blue at the N-terminal end. The β-flap, catalytic site, and visible InsP6-interacting side chains are shown as sticks. Data are expressed as mean ± SD. ND, not determinable. (B) Western blot analysis of RTX in supernatant harvested from log-phase V. cholerae cultures. Supernatants from V. cholerae strains harboring either an intact rtxA gene (wt), a null mutation in rtxArtxA), or point mutations in the region encoding the CPD domain of RTX (C140A is catalytic-dead; R182Q/K183N is mutated at two InsP6-binding residues; and W192A is a β-flap mutation) were blotted using an anti-CPD antibody. (C) Actin crosslinking induced upon incubation of V. cholerae with HFF cells. V. cholerae strains used in (A) were incubated with HFFs for 90 min, then the HFF cells were lysed. Actin crosslinking was visualized by SDS-PAGE and Western blotting by using an actin-specific antibody. The crosslinked forms of actin are labeled to the right.

We sought to determine if the mutation of non–InsP6-interacting residues in the β-flap might “decouple” InsP6-binding affinity from the autocatalytic activity of the enzyme. We focused on W192 in strand G3, which makes van der Waals contacts with strands G1 and G2 (Fig. 3A), and D178, which stabilizes the G1-G2 β-hairpin by hydrogen bonding with three backbone amide nitrogens and helps anchor the center of the G2 strand by forming a salt bridge with the side chain of H184 (Fig. 3A). We made conservative (W192F and D178N) and more potent (W192A and D178A) mutations; as controls, we also mutated nearby solvent-facing residues D191 and E179 (D191A and E179A). The conservative mutations (W192F and D178N) minimally altered InsP6-binding affinity yet considerably reduced autocatalysis relative to the wild type and controls D191A and E179A (Fig. 3A, right). Furthermore, the more potent W192A and D178A mutations induced only a modest drop in InsP6-binding affinity yet a more severe defect in catalysis. The D178A mutant exhibited InsP6 binding and catalytic activity similar to those of the R182Q mutant, consistent with their similar positions in the β-flap. The W192A mutant was most dramatically affected; despite having moderate affinity for InsP6 (9.3 ± 0.6 μM), W192A had a catalytic defect that was twice as strong as that of the InsP6-binding mutant K195N. Thus, mutation of residues that apparently communicate InsP6 binding to the active site through structural rearrangement of the β-flap can decouple InsP6 binding from enzyme activation. The proposed structural rearrangement, however, appears to be subtle because the circular dichroism (CD) spectra of the free- and InsP6-bound enzymes were nearly superimposable (fig. S13).

To assess the biological importance of InsP6 binding and β-flap integrity on RTX CPD function in vivo, we constructed V. cholerae strains containing CPD point mutations C140A (catalytic dead), R182Q/K183N (InsP6-binding defective), and W192A (β-flap–transition defective). Western detection of the CPD (Fig. 3B) showed that although most wild-type RTX was autoprocessed during the growth of V. cholerae in LB medium, RTX containing catalytic dead (C140A), InsP6-binding (R182Q/K183N), or β-flap (W192A) mutations were unprocessed (Fig. 3B). Thus, even in the presence of high levels of InsP6 in LB medium, all three mutations prevented CPD activation in the native RTX. When V. cholerae mutant strains or supernatants were incubated with human foreskin fibroblast (HFF) cells, intracellular actin cross-linking induced by the mutant strains R182Q/K183N and W192A was severely reduced relative to the wild type, whereas C140A failed to induce actin cross-linking (Fig. 3C and fig. S14). Thus, InsP6 binding and an intact β-flap are required for RTX autocleavage and effector function.

The InsP6-interacting residues in related MARTX cysteine protease domains are almost invariably conserved (fig. S2), which strongly suggests a shared mechanism for toxin activation in Gram-negative bacteria. Because InsP6 is exclusive to eukaryotes (15) and is present at cytosolic concentrations >10 μM (16), the responsiveness of the MARTX family to InsP6 through the evolution of a proteolytic biosensor seems an ingenious strategy for regulating the function of a secreted toxin.

Supporting Online Material

www.sciencemag.org/cgi/content/full/322/5899/265/DC1

Materials and Methods

Figs. S1 to S14

Tables S1 to S3

References

References and Notes

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