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Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins

Nature Structural & Molecular Biology volume 18, pages 364–371 (2011)Cite this article

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Abstract

An internal cysteine protease domain (CPD) autoproteolytically regulates Clostridium difficile glucosylating toxins by releasing a cytotoxic effector domain into target cells. CPD activity is itself allosterically regulated by the eukaryote-specific molecule inositol hexakisphosphate (InsP6). Although allostery controls the function of most proteins, the molecular details underlying this regulatory mechanism are often difficult to characterize. Here we use chemical probes to show that apo-CPD is in dynamic equilibrium between active and inactive states. InsP6 markedly shifts this equilibrium toward an active conformer that is further restrained upon binding a suicide substrate. Structural analyses combined with systematic mutational and disulfide bond engineering studies show that residues within a β-hairpin region functionally couple the InsP6-binding site to the active site. Collectively, our results identify an allosteric circuit that allows bacterial virulence factors to sense and respond to the eukaryotic environment.

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Figure 1: Development of an activity-based probe assay for measuring TcdB CPD activity.
Figure 2: Detection of an active conformer in the apo state using an activity-based probe.
Figure 3: Effect of InsP6 and P1 leucine inhibitor modification on TcdB CPD conformational mobility.
Figure 4: Structure of TcdB CPD and comparison with TcdA CPD.
Figure 5: InsP6 binding induces movement of conserved tryptophan in β-flap region.
Figure 6: Effect of redox state on InsP6 responsiveness of engineered disulfide bond mutants in the β-flap.

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Acknowledgements

We thank J. Wells for helpful suggestions in the preparation of this manuscript. A.S. is supported by a US National Institutes of Health (NIH) National Institutes of General Medical Sciences grant (K99GM092934). P.J.L. is a Damon Runyon Fellow, supported by the Damon Runyon Cancer Research Foundation. A.W.P. is supported by a US National Science Foundation Graduate Research Fellowship. K.C.G. is supported by the Howard Hughes Medical Institute and NIH grant NIH-RO1AI48540. M.B. is supported by the Burroughs Wellcome Foundation and NIH grants R01EB005011 and R01AI078947.

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Author notes

  1. Patrick J Lupardus & Victoria E Albrow

    Present address: Current addresses: Genentech, South San Francisco, California, USA (P.J.L.); Pfizer, Sandwich, UK (V.E.A.).,

  2. ashen2@stanford.edu

Authors and Affiliations

  1. Department of Pathology, Stanford School of Medicine, Stanford, California, USA

    Aimee Shen, Victoria E Albrow & Matthew Bogyo

  2. Department of Molecular and Cellular Physiology, Stanford School of Medicine, Stanford, California, USA

    Patrick J Lupardus & K Christopher Garcia

  3. Howard Hughes Institute, Stanford School of Medicine, Stanford, California, USA

    Patrick J Lupardus & K Christopher Garcia

  4. Department of Chemical and Systems Biology, Stanford School of Medicine, Stanford, California, USA

    Malte M Gersch

  5. Department of Chemistry, Center for Integrated Protein Science Munich (CIPSM), Technische Universitat Munchen, Garching, Germany

    Aaron W Puri

  6. Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, California, USA

    Matthew Bogyo

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Contributions

A.S. developed and executed autocleavage, probe labeling, limited proteolysis and tryptophan fluorescence assays; constructed, purified and expressed CPD variants; prepared CPD for crystallization studies; helped design the AWP19 probe; and wrote the manuscript. P.J.L. crystallized InsP6-bound TcdB CPD, collected the data, solved and analyzed the structure, generated Figure 4 and Table 1 and provided advice on the manuscript. M.M.G. synthesized AWP19, helped with initial characterization of TcdB CPD mutants using a fluorogenic substrate assay and the probe labeling assay and provided creative input. A.W.P. helped design and synthesize the AWP19 probe and performed initial characterization of probe labeling of TcdB CPD. V.E.A. helped design the AWP19 probe and provided advice on probe usage. K.C.G. provided advice on the manuscript and provided financial support. M.B. provided creative input and financial support, and helped write the manuscript.

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Correspondence to Matthew Bogyo.

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Shen, A., Lupardus, P., Gersch, M. et al. Defining an allosteric circuit in the cysteine protease domain of Clostridium difficile toxins. Nat Struct Mol Biol 18, 364–371 (2011). https://doi.org/10.1038/nsmb.1990

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