Abstract
MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-Å structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.
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Acknowledgements
We thank P. Gulig (University of Florida) for generously providing Vibrio vulnificus CMCP6 genomic DNA, E. Shank and R. Kolter (Harvard Medical School) for providing the Photorhabdus luminescens TTO1 strain, and M. Blokech and G. Schoolnik (Stanford School of Medicine) for help with V. cholerae strain construction and for providing V. cholerae genomic DNA. P.J.L. is supported by the Damon Runyon Cancer Research Foundation. K.C.G. is supported by the Keck Foundation and the Howard Hughes Medical Institute. M.B. is supported by the Burroughs Wellcome Foundation, the Searle Scholars Program, the US National Institutes of Health National Technology Center for Networks and Pathways (grant U54-RR020843) and the Human Frontier Science Program (grant RGP0024/2006-C).
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The inhibitor screen, synthesis of AS01, AS02 and AS04, protein expression and purification, cleavage assays, FT-MS data analysis, V. cholerae strain construction, actin crosslinking assays, MARTXVc silver staining and western blot analyses were performed by A.S. Crystallization of the MARTX CPD–InsP6–JCP598 complex was performed by A.S. and P.J.L. P.J.L. collected the data, solved and analyzed the structure, and generated the figures of the inhibitor-bound CPD structure. J.C.P. provided the cysteine protease compound library. V.E.A. synthesized JCP598, AS01 and VEA223, guided A.S. in the synthesis of AS01, AS02 and AS04, and assessed the integrity of all compounds described in this paper. A.G. designed the conditions for running samples for FT-MS, ran the samples for FT-MS and provided advice in FT-MS analysis. Creative input and financial support for the project were provided by M.B. The manuscript was written by A.S. and M.B. with advice from P.J.L., V.E.A., K.C.G. and A.G.
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Shen, A., Lupardus, P., Albrow, V. et al. Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin. Nat Chem Biol 5, 469–478 (2009). https://doi.org/10.1038/nchembio.178
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DOI: https://doi.org/10.1038/nchembio.178
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