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Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

2009; Elsevier BV; Volume: 284; Issue: 39 Linguagem: Inglês

10.1074/jbc.m109.025510

1083-351X

Kateřina Procházková, L. Shuvalova, G. Minasov, Zdeněk Vobůrka, W.F. Anderson, K.J.F. Satchell,

Yersinia bacterium, plague, ectoparasites research

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP6). In this study, we demonstrated that InsP6 is not simply an allosteric cofactor, but rather binding of InsP6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP6-bound unprocessed form of CPD was determined and revealed the scissile bond Leu3428–Ala3429 captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP6, but was reactivated for high affinity binding of InsP6 by cooperative binding of both a new substrate and InsP6. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease. The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP6). In this study, we demonstrated that InsP6 is not simply an allosteric cofactor, but rather binding of InsP6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP6-bound unprocessed form of CPD was determined and revealed the scissile bond Leu3428–Ala3429 captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP6, but was reactivated for high affinity binding of InsP6 by cooperative binding of both a new substrate and InsP6. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease. Multifunctional-autoprocessing repeats-in-toxin (MARTX) 3The abbreviations used are: MARTXmultifunctional-autoprocessing repeats in toxinCPDcysteine protease domainACDactin cross-linking domainRIDRhoGTPase inactivation domainNEMN-ethylmaleimideCKchloromethyl ketoneTEVtobacco etch virusITCisothermal titration calorimetryFT-MSFourier transform mass spectrometryInsP6inositol hexakisphosphaterCPDrecombinant CPD. 3The abbreviations used are: MARTXmultifunctional-autoprocessing repeats in toxinCPDcysteine protease domainACDactin cross-linking domainRIDRhoGTPase inactivation domainNEMN-ethylmaleimideCKchloromethyl ketoneTEVtobacco etch virusITCisothermal titration calorimetryFT-MSFourier transform mass spectrometryInsP6inositol hexakisphosphaterCPDrecombinant CPD. toxins are a family of large bacterial protein toxins with conserved repeat regions at the N and C termini that are predicted to transfer effector domains located between the repeats across the eukaryotic cell plasma membrane (1Satchell K.J. Infect. Immun. 2007; 75: 5079-5084Crossref PubMed Scopus (90) Google Scholar). The best characterized MARTX is the >450-kDa secreted virulence-associated MARTX of Vibrio cholerae. This toxin causes disassembly of the actin cytoskeleton and enhances V. cholerae colonization of the small intestine, possibly by facilitating evasion of phagocytic cells (2Olivier V. Haines 3rd, G.K. Tan Y. Satchell K.J. Infect. Immun. 2007; 75: 5035-5042Crossref PubMed Scopus (73) Google Scholar, 3Olivier V. Salzman N.H. Satchell K.J. Infect. Immun. 2007; 75: 5043-5051Crossref PubMed Scopus (67) Google Scholar). The central region of the V. cholerae MARTX toxin contains four discrete domains: the actin cross-linking domain (ACD) that introduces lysine-glutamate cross-links between actin protomers (4Kudryashov D.S. Durer Z.A. Ytterberg A.J. Sawaya M.R. Pashkov I. Prochazkova K. Yeates T.O. Loo R.R. Loo J.A. Satchell K.J. Reisler E. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18537-18542Crossref PubMed Scopus (55) Google Scholar, 5Sheahan K.L. Cordero C.L. Satchell K.J. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9798-9803Crossref PubMed Scopus (113) Google Scholar), the Rho-inactivating domain (RID) that disables small Rho GTPases (6Sheahan K.L. Satchell K.J. Cell. Microbiol. 2007; 9: 1324-1335Crossref PubMed Scopus (65) Google Scholar), an αβ hydrolase of unknown function (1Satchell K.J. Infect. Immun. 2007; 75: 5079-5084Crossref PubMed Scopus (90) Google Scholar), and an autoprocessing cysteine protease domain (CPD) (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar).The CPD is a 25-kDa domain found in all MARTX toxins located just before the start of the C-terminal repeats (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar). This domain is activated for autoproteolysis upon binding inositol hexakisphosphate (InsP6) (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), a molecule ubiquitously present in eukaryotic cell cytosol (9French P.J. Bunce C.M. Stephens L.R. Lord J.M. McConnell F.M. Brown G. Creba J.A. Michell R.H. Proc. Biol. Sci. 1991; 245: 193-201Crossref PubMed Scopus (56) Google Scholar, 10Jackson T.R. Hallam T.J. Downes C.P. Hanley M.R. EMBO J. 1987; 6: 49-54Crossref PubMed Scopus (90) Google Scholar, 11Vallejo M. Jackson T. Lightman S. Hanley M.R. Nature. 1987; 330: 656-658Crossref PubMed Scopus (133) Google Scholar), but absent in extracellular spaces and bacteria. Thus, autocatalytic processing would not occur until after translocation of the CPD and effector domains is completed. In the context of the holotoxin, catalytic residue Cys3568 was found to be essential for the toxin to induce efficient actin cross-linking by the ACD and Rho inactivation by the RID, demonstrating that autoprocessing is essential for MARTX to induce cell rounding (8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar).While it is clear that InsP6 activates the CPD and that autoprocessing is essential for MARTX function (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the mechanism by which InsP6 activates CPD is not well understood. Furthermore, only one processing site at Leu3428–Ala3429 has been identified, although multiple processing events would be required to release each effector independently. In fact, after autoprocessing at Leu3428–Ala3429, CPD is reported to adopt a conformation with reduced affinity for InsP6 (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), raising questions as to how the protease might process MARTX at other sites.We present here the structure of the pre-processed form of the V. cholerae MARTX CPD bound to InsP6. Our results demonstrate that autoprocessing is activated by rearrangement of a β-hairpin loop upon InsP6 binding that locks the N terminus of the CPD in the active site, facilitating hydrolysis of the Leu3428–Ala3429 peptide bond. After autoprocessing, CPD adopts a post-processing form that has poor affinity for InsP6 and thus must be cooperatively reactivated for high affinity binding of InsP6 by association of a new substrate. As a consequence, we are able to demonstrate how CPD cleaves MARTX toxin between effector domains and releases them from the large toxin resulting in increased catalytic activity of the effectors.DISCUSSIONIn this study, we determined the crystal structure of the enzyme-substrate complex of V. cholerae MARTX CPD and used the structural information to suggest biochemical experiments that probe the site and mechanism for InsP6-induced autoproteolysis both at Leu3428–Ala3429 and at other sites of the MARTX toxin. The results support a model of controlled activation in which InsP6 is important for stabilization of the pro-enzyme prior to autoprocessing specifically at leucine residues, and secondly, functioning as a cooperative factor along with a new substrate for reactivation to process other sites. The subsequent autoprocessing would release the effector domains ACD, RID, and αβ hydrolase to independently access different targets. The final result will be actin destruction due to cross-linking of cytosol localized monomeric G-actin by the ACD and membrane localized Rho GTPases by the RID. Consistent with the important role of autoprocessing, both in vitro (Fig. 7) and in vivo (8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar), the ability of MARTX to efficiently cross-link actin was dependent upon it first being autoprocessed to release the ACD as an independent domain.In this study, we sought to more clearly understand the mechanism of InsP6-induced autoprocessing. The key structure for coordination of the interdependence between the peptide to be processed and InsP6 was identified as a β-hairpin structure (β8β9) that contacts the target peptide through interaction of hydrophobic side chains on β8 with P1 Leu3428 and with InsP6 through arginine and lysine side chains on β9. Prior to InsP6 binding, the protein exists in a conformation that is trypsin-sensitive, most notably in a portion that may coordinate proper alignment of the catalytic cysteine. Upon InsP6 binding, the β-flap undergoes a structural alteration that locks the substrate in the S1 pocket in a rigid structure amenable to substrate-activated processing. Subsequent to autoprocessing, the β-flap may shift resulting in release of InsP6, although cooperative binding of a new substrate and InsP6 results in reactivation.Previously, a role for the β-flap in coupling binding with activation was postulated and Asp3606 and Trp3620 were identified as residues within the β-flap that would affect folding and thereby establish contact with the protein core (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). In this work, the key structure that is altered is identified as β8β9 by partial trypsin digestion.This model for activation of CPD by formation of a stable enzyme-substrate complex is quite distinct from a recently proposed model for activation by an allosteric structural conversion to expose a previously buried catalytic cysteine to substrate (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). That allosteric mechanism was suggested when it was observed that a 1-min exposure of pro-CPD to NEM followed by addition of InsP6, poorly inhibited autoprocessing, initially suggesting the catalytic cysteine is buried, a result we confirm in Fig. 4C. However, we found that a longer incubation of pro-CPD or RtxA1580–3909 with NEM fully inhibited processing indicating transient exposure of the cysteine (Figs. 5 and 7). Inspection of the pre-processed structure suggests the N terminus frequently, if not predominantly, occupies the S1 site, thus, inhibition by NEM occurs only over time, targeting the cysteine only when the N terminus transiently departs the active site. This time dependence was reduced by increasing temperature to promote more dynamic motion of the N terminus and, most definitively, by complete removal of the N terminus by autoprocessing. The free accessibility of the cysteine in the processed form of CPD is also strongly supported by the observation that a fluorescent maleimide reacts with the cysteine of post-CPD, but not the pre-processed form, when it is added to a CPD autoprocessing assay (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar).The previously proposed allosteric mechanism was further suggested by the observation that autoprocessing of pro-CPD was inhibited when exposed to NEM and InsP6 simultaneously (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). These data directly conflict with our results that simultaneous addition of NEM with 100 μm InsP6 resulted in a protein that was ∼80% processed (Fig. 4D). It is notable that the experiment cited as supporting a structural transition was carried out with pro-CPD that had previously been exposed to NEM for 1 min, a treatment expected to inhibit ∼50% of CPD (Fig. 4C). Thus, the pro-CPD in that experiment was probably inactivated prior to NEM and InsP6 co-injection, preventing autoprocessing. Thus, all data support a cooperative mechanism for activation, as opposed to the allosteric structural transition model, for pro-enzyme activation and reactivation both in the context of small recombinant CPD proteins (Fig. 6) and the larger recombinant protein RtxA1580–3909 (Fig. 7).In the context of the MARTX holotoxin, we propose that translocation of the ∼185-kDa central region of MARTX to the cytosol likely involves at least partial unfolding of the toxin, because translocation of a fully folded protein would require a substantial pore and it has been demonstrated that there is no leakage of cytosol contents due to MARTX (37Fullner K.J. Mekalanos J.J. EMBO J. 2000; 19: 5315-5323Crossref PubMed Scopus (136) Google Scholar). Upon CPD translocation, Leu3428 moves into the CPD S1 site facilitating high affinity binding of InsP6. Upon binding, β8β9 adopts the proper conformation locking the scissile bond into the active site where it is cleaved. Reactivation of CPD then occurs by reoccupation of the S1 site by a new substrate and binding of InsP6. This reactivation results in cleavage at other leucine residues located between the effector domains releasing them from the larger toxin. Secondary structure predictions have shown that these leucines are present in unstructured regions that flank the effectors, explaining the specificity of preferred cleavage sites despite the fact that CPD is apparently a promiscuous protease able to process at almost any exposed leucine. Interestingly, autoprocessing of RtxA1580–3909 by its own CPD was more efficient than in trans processing by pro-CPD (Fig. 7A). This result suggests that close proximity to other processing sites may create the necessary high local concentration of substrate facilitating immediate reoccupation of the active site after departure of Leu3428–Ala3429. As an extension, this observation might indicate that the effector domains are associated with CPD until processing is completed.The specificity of this enzyme for leucine is of keen interest. All Clan CD proteases are recognized for having high specificity for processing: for example, eukaryotic caspases at Asp residues, the gingipain K and R proteases of Porphyromonas gingivalis at Arg and Lys, respectively, and mammalian legumain at Asn (29Chen J.M. Rawlings N.D. Stevens R.A. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar, 30Rawlings N.D. Morton F.R. Kok C.Y. Kong J. Barrett A.J. Nucleic Acids Res. 2008; 36: D320-D325Crossref PubMed Scopus (509) Google Scholar). The identification of specificity for Leu of the MARTX CPD indicates that this and similar proteases represent a unique family of proteases, although the structure of the catalytic site clearly places it in Clan CD. All the processing sites defined in this study are shown in Fig. 7F in the context of 10 amino acids on each side of the scissile bond. By analyzing these peptides, no conservation of residues other than Leu in P1 site is evident, and mutation analysis indicated any of the two neighboring residues can be altered. Yet, among the preferred processing sites, there is a preference for a small amino acid residue on each side of the Leu (Fig. 7F). Extension of this analysis to the Ala-Leu-Gly or Ser-Leu-Gly recognition sites for Clostridium difficile Toxin B further indicates preference for small-Leu-small suggesting a conserved preference among this family of proteases (38Rupnik M. Pabst S. Rupnik M. von Eichel-Streiber C. Urlaub H. Söoling H.D. Microbiology. 2005; 151: 199-208Crossref PubMed Scopus (83) Google Scholar).As a conclusion, we have described a new model for delivery of multiple toxin effectors where, upon translocation of the central region into eukaryotic cells, CPD binds InsP6, cleaves itself and then other sites in MARTX, releasing the functional domains that then reach their targets and contribute to pathogenesis of cholera. Multifunctional-autoprocessing repeats-in-toxin (MARTX) 3The abbreviations used are: MARTXmultifunctional-autoprocessing repeats in toxinCPDcysteine protease domainACDactin cross-linking domainRIDRhoGTPase inactivation domainNEMN-ethylmaleimideCKchloromethyl ketoneTEVtobacco etch virusITCisothermal titration calorimetryFT-MSFourier transform mass spectrometryInsP6inositol hexakisphosphaterCPDrecombinant CPD. 3The abbreviations used are: MARTXmultifunctional-autoprocessing repeats in toxinCPDcysteine protease domainACDactin cross-linking domainRIDRhoGTPase inactivation domainNEMN-ethylmaleimideCKchloromethyl ketoneTEVtobacco etch virusITCisothermal titration calorimetryFT-MSFourier transform mass spectrometryInsP6inositol hexakisphosphaterCPDrecombinant CPD. toxins are a family of large bacterial protein toxins with conserved repeat regions at the N and C termini that are predicted to transfer effector domains located between the repeats across the eukaryotic cell plasma membrane (1Satchell K.J. Infect. Immun. 2007; 75: 5079-5084Crossref PubMed Scopus (90) Google Scholar). The best characterized MARTX is the >450-kDa secreted virulence-associated MARTX of Vibrio cholerae. This toxin causes disassembly of the actin cytoskeleton and enhances V. cholerae colonization of the small intestine, possibly by facilitating evasion of phagocytic cells (2Olivier V. Haines 3rd, G.K. Tan Y. Satchell K.J. Infect. Immun. 2007; 75: 5035-5042Crossref PubMed Scopus (73) Google Scholar, 3Olivier V. Salzman N.H. Satchell K.J. Infect. Immun. 2007; 75: 5043-5051Crossref PubMed Scopus (67) Google Scholar). The central region of the V. cholerae MARTX toxin contains four discrete domains: the actin cross-linking domain (ACD) that introduces lysine-glutamate cross-links between actin protomers (4Kudryashov D.S. Durer Z.A. Ytterberg A.J. Sawaya M.R. Pashkov I. Prochazkova K. Yeates T.O. Loo R.R. Loo J.A. Satchell K.J. Reisler E. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18537-18542Crossref PubMed Scopus (55) Google Scholar, 5Sheahan K.L. Cordero C.L. Satchell K.J. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9798-9803Crossref PubMed Scopus (113) Google Scholar), the Rho-inactivating domain (RID) that disables small Rho GTPases (6Sheahan K.L. Satchell K.J. Cell. Microbiol. 2007; 9: 1324-1335Crossref PubMed Scopus (65) Google Scholar), an αβ hydrolase of unknown function (1Satchell K.J. Infect. Immun. 2007; 75: 5079-5084Crossref PubMed Scopus (90) Google Scholar), and an autoprocessing cysteine protease domain (CPD) (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar). multifunctional-autoprocessing repeats in toxin cysteine protease domain actin cross-linking domain RhoGTPase inactivation domain N-ethylmaleimide chloromethyl ketone tobacco etch virus isothermal titration calorimetry Fourier transform mass spectrometry inositol hexakisphosphate recombinant CPD. multifunctional-autoprocessing repeats in toxin cysteine protease domain actin cross-linking domain RhoGTPase inactivation domain N-ethylmaleimide chloromethyl ketone tobacco etch virus isothermal titration calorimetry Fourier transform mass spectrometry inositol hexakisphosphate recombinant CPD. The CPD is a 25-kDa domain found in all MARTX toxins located just before the start of the C-terminal repeats (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar). This domain is activated for autoproteolysis upon binding inositol hexakisphosphate (InsP6) (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), a molecule ubiquitously present in eukaryotic cell cytosol (9French P.J. Bunce C.M. Stephens L.R. Lord J.M. McConnell F.M. Brown G. Creba J.A. Michell R.H. Proc. Biol. Sci. 1991; 245: 193-201Crossref PubMed Scopus (56) Google Scholar, 10Jackson T.R. Hallam T.J. Downes C.P. Hanley M.R. EMBO J. 1987; 6: 49-54Crossref PubMed Scopus (90) Google Scholar, 11Vallejo M. Jackson T. Lightman S. Hanley M.R. Nature. 1987; 330: 656-658Crossref PubMed Scopus (133) Google Scholar), but absent in extracellular spaces and bacteria. Thus, autocatalytic processing would not occur until after translocation of the CPD and effector domains is completed. In the context of the holotoxin, catalytic residue Cys3568 was found to be essential for the toxin to induce efficient actin cross-linking by the ACD and Rho inactivation by the RID, demonstrating that autoprocessing is essential for MARTX to induce cell rounding (8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar). While it is clear that InsP6 activates the CPD and that autoprocessing is essential for MARTX function (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the mechanism by which InsP6 activates CPD is not well understood. Furthermore, only one processing site at Leu3428–Ala3429 has been identified, although multiple processing events would be required to release each effector independently. In fact, after autoprocessing at Leu3428–Ala3429, CPD is reported to adopt a conformation with reduced affinity for InsP6 (7Prochazkova K. Satchell K.J. J. Biol. Chem. 2008; 283: 23656-23664Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), raising questions as to how the protease might process MARTX at other sites. We present here the structure of the pre-processed form of the V. cholerae MARTX CPD bound to InsP6. Our results demonstrate that autoprocessing is activated by rearrangement of a β-hairpin loop upon InsP6 binding that locks the N terminus of the CPD in the active site, facilitating hydrolysis of the Leu3428–Ala3429 peptide bond. After autoprocessing, CPD adopts a post-processing form that has poor affinity for InsP6 and thus must be cooperatively reactivated for high affinity binding of InsP6 by association of a new substrate. As a consequence, we are able to demonstrate how CPD cleaves MARTX toxin between effector domains and releases them from the large toxin resulting in increased catalytic activity of the effectors. DISCUSSIONIn this study, we determined the crystal structure of the enzyme-substrate complex of V. cholerae MARTX CPD and used the structural information to suggest biochemical experiments that probe the site and mechanism for InsP6-induced autoproteolysis both at Leu3428–Ala3429 and at other sites of the MARTX toxin. The results support a model of controlled activation in which InsP6 is important for stabilization of the pro-enzyme prior to autoprocessing specifically at leucine residues, and secondly, functioning as a cooperative factor along with a new substrate for reactivation to process other sites. The subsequent autoprocessing would release the effector domains ACD, RID, and αβ hydrolase to independently access different targets. The final result will be actin destruction due to cross-linking of cytosol localized monomeric G-actin by the ACD and membrane localized Rho GTPases by the RID. Consistent with the important role of autoprocessing, both in vitro (Fig. 7) and in vivo (8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar), the ability of MARTX to efficiently cross-link actin was dependent upon it first being autoprocessed to release the ACD as an independent domain.In this study, we sought to more clearly understand the mechanism of InsP6-induced autoprocessing. The key structure for coordination of the interdependence between the peptide to be processed and InsP6 was identified as a β-hairpin structure (β8β9) that contacts the target peptide through interaction of hydrophobic side chains on β8 with P1 Leu3428 and with InsP6 through arginine and lysine side chains on β9. Prior to InsP6 binding, the protein exists in a conformation that is trypsin-sensitive, most notably in a portion that may coordinate proper alignment of the catalytic cysteine. Upon InsP6 binding, the β-flap undergoes a structural alteration that locks the substrate in the S1 pocket in a rigid structure amenable to substrate-activated processing. Subsequent to autoprocessing, the β-flap may shift resulting in release of InsP6, although cooperative binding of a new substrate and InsP6 results in reactivation.Previously, a role for the β-flap in coupling binding with activation was postulated and Asp3606 and Trp3620 were identified as residues within the β-flap that would affect folding and thereby establish contact with the protein core (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). In this work, the key structure that is altered is identified as β8β9 by partial trypsin digestion.This model for activation of CPD by formation of a stable enzyme-substrate complex is quite distinct from a recently proposed model for activation by an allosteric structural conversion to expose a previously buried catalytic cysteine to substrate (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). That allosteric mechanism was suggested when it was observed that a 1-min exposure of pro-CPD to NEM followed by addition of InsP6, poorly inhibited autoprocessing, initially suggesting the catalytic cysteine is buried, a result we confirm in Fig. 4C. However, we found that a longer incubation of pro-CPD or RtxA1580–3909 with NEM fully inhibited processing indicating transient exposure of the cysteine (Figs. 5 and 7). Inspection of the pre-processed structure suggests the N terminus frequently, if not predominantly, occupies the S1 site, thus, inhibition by NEM occurs only over time, targeting the cysteine only when the N terminus transiently departs the active site. This time dependence was reduced by increasing temperature to promote more dynamic motion of the N terminus and, most definitively, by complete removal of the N terminus by autoprocessing. The free accessibility of the cysteine in the processed form of CPD is also strongly supported by the observation that a fluorescent maleimide reacts with the cysteine of post-CPD, but not the pre-processed form, when it is added to a CPD autoprocessing assay (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar).The previously proposed allosteric mechanism was further suggested by the observation that autoprocessing of pro-CPD was inhibited when exposed to NEM and InsP6 simultaneously (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). These data directly conflict with our results that simultaneous addition of NEM with 100 μm InsP6 resulted in a protein that was ∼80% processed (Fig. 4D). It is notable that the experiment cited as supporting a structural transition was carried out with pro-CPD that had previously been exposed to NEM for 1 min, a treatment expected to inhibit ∼50% of CPD (Fig. 4C). Thus, the pro-CPD in that experiment was probably inactivated prior to NEM and InsP6 co-injection, preventing autoprocessing. Thus, all data support a cooperative mechanism for activation, as opposed to the allosteric structural transition model, for pro-enzyme activation and reactivation both in the context of small recombinant CPD proteins (Fig. 6) and the larger recombinant protein RtxA1580–3909 (Fig. 7).In the context of the MARTX holotoxin, we propose that translocation of the ∼185-kDa central region of MARTX to the cytosol likely involves at least partial unfolding of the toxin, because translocation of a fully folded protein would require a substantial pore and it has been demonstrated that there is no leakage of cytosol contents due to MARTX (37Fullner K.J. Mekalanos J.J. EMBO J. 2000; 19: 5315-5323Crossref PubMed Scopus (136) Google Scholar). Upon CPD translocation, Leu3428 moves into the CPD S1 site facilitating high affinity binding of InsP6. Upon binding, β8β9 adopts the proper conformation locking the scissile bond into the active site where it is cleaved. Reactivation of CPD then occurs by reoccupation of the S1 site by a new substrate and binding of InsP6. This reactivation results in cleavage at other leucine residues located between the effector domains releasing them from the larger toxin. Secondary structure predictions have shown that these leucines are present in unstructured regions that flank the effectors, explaining the specificity of preferred cleavage sites despite the fact that CPD is apparently a promiscuous protease able to process at almost any exposed leucine. Interestingly, autoprocessing of RtxA1580–3909 by its own CPD was more efficient than in trans processing by pro-CPD (Fig. 7A). This result suggests that close proximity to other processing sites may create the necessary high local concentration of substrate facilitating immediate reoccupation of the active site after departure of Leu3428–Ala3429. As an extension, this observation might indicate that the effector domains are associated with CPD until processing is completed.The specificity of this enzyme for leucine is of keen interest. All Clan CD proteases are recognized for having high specificity for processing: for example, eukaryotic caspases at Asp residues, the gingipain K and R proteases of Porphyromonas gingivalis at Arg and Lys, respectively, and mammalian legumain at Asn (29Chen J.M. Rawlings N.D. Stevens R.A. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar, 30Rawlings N.D. Morton F.R. Kok C.Y. Kong J. Barrett A.J. Nucleic Acids Res. 2008; 36: D320-D325Crossref PubMed Scopus (509) Google Scholar). The identification of specificity for Leu of the MARTX CPD indicates that this and similar proteases represent a unique family of proteases, although the structure of the catalytic site clearly places it in Clan CD. All the processing sites defined in this study are shown in Fig. 7F in the context of 10 amino acids on each side of the scissile bond. By analyzing these peptides, no conservation of residues other than Leu in P1 site is evident, and mutation analysis indicated any of the two neighboring residues can be altered. Yet, among the preferred processing sites, there is a preference for a small amino acid residue on each side of the Leu (Fig. 7F). Extension of this analysis to the Ala-Leu-Gly or Ser-Leu-Gly recognition sites for Clostridium difficile Toxin B further indicates preference for small-Leu-small suggesting a conserved preference among this family of proteases (38Rupnik M. Pabst S. Rupnik M. von Eichel-Streiber C. Urlaub H. Söoling H.D. Microbiology. 2005; 151: 199-208Crossref PubMed Scopus (83) Google Scholar).As a conclusion, we have described a new model for delivery of multiple toxin effectors where, upon translocation of the central region into eukaryotic cells, CPD binds InsP6, cleaves itself and then other sites in MARTX, releasing the functional domains that then reach their targets and contribute to pathogenesis of cholera. In this study, we determined the crystal structure of the enzyme-substrate complex of V. cholerae MARTX CPD and used the structural information to suggest biochemical experiments that probe the site and mechanism for InsP6-induced autoproteolysis both at Leu3428–Ala3429 and at other sites of the MARTX toxin. The results support a model of controlled activation in which InsP6 is important for stabilization of the pro-enzyme prior to autoprocessing specifically at leucine residues, and secondly, functioning as a cooperative factor along with a new substrate for reactivation to process other sites. The subsequent autoprocessing would release the effector domains ACD, RID, and αβ hydrolase to independently access different targets. The final result will be actin destruction due to cross-linking of cytosol localized monomeric G-actin by the ACD and membrane localized Rho GTPases by the RID. Consistent with the important role of autoprocessing, both in vitro (Fig. 7) and in vivo (8Sheahan K.L. Cordero C.L. Satchell K.J. EMBO J. 2007; 26: 2552-2561Crossref PubMed Scopus (78) Google Scholar), the ability of MARTX to efficiently cross-link actin was dependent upon it first being autoprocessed to release the ACD as an independent domain. In this study, we sought to more clearly understand the mechanism of InsP6-induced autoprocessing. The key structure for coordination of the interdependence between the peptide to be processed and InsP6 was identified as a β-hairpin structure (β8β9) that contacts the target peptide through interaction of hydrophobic side chains on β8 with P1 Leu3428 and with InsP6 through arginine and lysine side chains on β9. Prior to InsP6 binding, the protein exists in a conformation that is trypsin-sensitive, most notably in a portion that may coordinate proper alignment of the catalytic cysteine. Upon InsP6 binding, the β-flap undergoes a structural alteration that locks the substrate in the S1 pocket in a rigid structure amenable to substrate-activated processing. Subsequent to autoprocessing, the β-flap may shift resulting in release of InsP6, although cooperative binding of a new substrate and InsP6 results in reactivation. Previously, a role for the β-flap in coupling binding with activation was postulated and Asp3606 and Trp3620 were identified as residues within the β-flap that would affect folding and thereby establish contact with the protein core (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). In this work, the key structure that is altered is identified as β8β9 by partial trypsin digestion. This model for activation of CPD by formation of a stable enzyme-substrate complex is quite distinct from a recently proposed model for activation by an allosteric structural conversion to expose a previously buried catalytic cysteine to substrate (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). That allosteric mechanism was suggested when it was observed that a 1-min exposure of pro-CPD to NEM followed by addition of InsP6, poorly inhibited autoprocessing, initially suggesting the catalytic cysteine is buried, a result we confirm in Fig. 4C. However, we found that a longer incubation of pro-CPD or RtxA1580–3909 with NEM fully inhibited processing indicating transient exposure of the cysteine (Figs. 5 and 7). Inspection of the pre-processed structure suggests the N terminus frequently, if not predominantly, occupies the S1 site, thus, inhibition by NEM occurs only over time, targeting the cysteine only when the N terminus transiently departs the active site. This time dependence was reduced by increasing temperature to promote more dynamic motion of the N terminus and, most definitively, by complete removal of the N terminus by autoprocessing. The free accessibility of the cysteine in the processed form of CPD is also strongly supported by the observation that a fluorescent maleimide reacts with the cysteine of post-CPD, but not the pre-processed form, when it is added to a CPD autoprocessing assay (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). The previously proposed allosteric mechanism was further suggested by the observation that autoprocessing of pro-CPD was inhibited when exposed to NEM and InsP6 simultaneously (25Lupardus P.J. Shen A. Bogyo M. Garcia K.C. Science. 2008; 322: 265-268Crossref PubMed Scopus (94) Google Scholar). These data directly conflict with our results that simultaneous addition of NEM with 100 μm InsP6 resulted in a protein that was ∼80% processed (Fig. 4D). It is notable that the experiment cited as supporting a structural transition was carried out with pro-CPD that had previously been exposed to NEM for 1 min, a treatment expected to inhibit ∼50% of CPD (Fig. 4C). Thus, the pro-CPD in that experiment was probably inactivated prior to NEM and InsP6 co-injection, preventing autoprocessing. Thus, all data support a cooperative mechanism for activation, as opposed to the allosteric structural transition model, for pro-enzyme activation and reactivation both in the context of small recombinant CPD proteins (Fig. 6) and the larger recombinant protein RtxA1580–3909 (Fig. 7). In the context of the MARTX holotoxin, we propose that translocation of the ∼185-kDa central region of MARTX to the cytosol likely involves at least partial unfolding of the toxin, because translocation of a fully folded protein would require a substantial pore and it has been demonstrated that there is no leakage of cytosol contents due to MARTX (37Fullner K.J. Mekalanos J.J. EMBO J. 2000; 19: 5315-5323Crossref PubMed Scopus (136) Google Scholar). Upon CPD translocation, Leu3428 moves into the CPD S1 site facilitating high affinity binding of InsP6. Upon binding, β8β9 adopts the proper conformation locking the scissile bond into the active site where it is cleaved. Reactivation of CPD then occurs by reoccupation of the S1 site by a new substrate and binding of InsP6. This reactivation results in cleavage at other leucine residues located between the effector domains releasing them from the larger toxin. Secondary structure predictions have shown that these leucines are present in unstructured regions that flank the effectors, explaining the specificity of preferred cleavage sites despite the fact that CPD is apparently a promiscuous protease able to process at almost any exposed leucine. Interestingly, autoprocessing of RtxA1580–3909 by its own CPD was more efficient than in trans processing by pro-CPD (Fig. 7A). This result suggests that close proximity to other processing sites may create the necessary high local concentration of substrate facilitating immediate reoccupation of the active site after departure of Leu3428–Ala3429. As an extension, this observation might indicate that the effector domains are associated with CPD until processing is completed. The specificity of this enzyme for leucine is of keen interest. All Clan CD proteases are recognized for having high specificity for processing: for example, eukaryotic caspases at Asp residues, the gingipain K and R proteases of Porphyromonas gingivalis at Arg and Lys, respectively, and mammalian legumain at Asn (29Chen J.M. Rawlings N.D. Stevens R.A. Barrett A.J. FEBS Lett. 1998; 441: 361-365Crossref PubMed Scopus (194) Google Scholar, 30Rawlings N.D. Morton F.R. Kok C.Y. Kong J. Barrett A.J. Nucleic Acids Res. 2008; 36: D320-D325Crossref PubMed Scopus (509) Google Scholar). The identification of specificity for Leu of the MARTX CPD indicates that this and similar proteases represent a unique family of proteases, although the structure of the catalytic site clearly places it in Clan CD. All the processing sites defined in this study are shown in Fig. 7F in the context of 10 amino acids on each side of the scissile bond. By analyzing these peptides, no conservation of residues other than Leu in P1 site is evident, and mutation analysis indicated any of the two neighboring residues can be altered. Yet, among the preferred processing sites, there is a preference for a small amino acid residue on each side of the Leu (Fig. 7F). Extension of this analysis to the Ala-Leu-Gly or Ser-Leu-Gly recognition sites for Clostridium difficile Toxin B further indicates preference for small-Leu-small suggesting a conserved preference among this family of proteases (38Rupnik M. Pabst S. Rupnik M. von Eichel-Streiber C. Urlaub H. Söoling H.D. Microbiology. 2005; 151: 199-208Crossref PubMed Scopus (83) Google Scholar). As a conclusion, we have described a new model for delivery of multiple toxin effectors where, upon translocation of the central region into eukaryotic cells, CPD binds InsP6, cleaves itself and then other sites in MARTX, releasing the functional domains that then reach their targets and contribute to pathogenesis of cholera. We thank R. Tungekar, B. Geissler, and C.-H. Luan for their input. We thankfully acknowledge the Keck Biophysics Facility at Northwestern University for use of ITC. Supplementary Material Download .pdf (1.15 MB) Help with pdf files Download .pdf (1.15 MB) Help with pdf files