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Structural and mechanistic basis of immunity toward endonuclease colicins

Nature Structural Biology volume 6, pages 243–252 (1999)Cite this article

Abstract

The crystal structure of the cytotoxic endonuclease domain from the bacterial toxin colicin E9 in complex with its cognate immunity protein Im9 reveals that the inhibitor does not bind at the active site, the core of which comprises the HNH motif found in intron-encoded homing endonucleases, but rather at an adjacent position leaving the active site exposed yet unable to bind DNA because of steric and electrostatic clashes with incoming substrate. Although its mode of action is unorthodox, Im9 is a remarkably effective inhibitor since it folds within milliseconds and then associates with its target endonuclease at the rate of diffusion to form an inactive complex with sub-femtomolar binding affinity. This hyperefficient mechanism of inhibition could be well suited to other toxic enzyme systems, particularly where the substrate is a polymer extending beyond the boundaries of the active site.

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Figure 1: The unfolding and refolding of Im9.
Figure 2: Im9 inhibits DNA and polyanion binding to the E9 DNase.
Figure 3: Structure of the E9 DNase–Im9 complex at 2.05 Å.
Figure 4: a, Topology diagram of the E9 DNase secondary-structure elements (with helices shown as red circles and ß-strands as green triangles) indicating the relative positions of the active site (shown in blue) and Im9 binding site (shown in yellow).
Figure 5: Stereo view of protein-protein interaction specificity residues that colocalize at the E9 DNase-Im9 interface.
Figure 6: Sequence and structure of the HNH homing endonuclease motif.
Figure 7: Im9 hinders access of double-stranded DNA into the E9 DNase active site.

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Acknowledgements

We thank the members of the UEA Colicin Research Group for valuable discussions, C. Garinot-Schneider (UEA) for early work on gel-shift experiments and A. Capaldi (Leeds) for help and advice regarding the folding experiments. We also thank the reviewers of this paper for their helpful comments. The work was generously supported by The Wellcome Trust and by the Biotechnology & Biological Sciences Research Council. N.F. was funded by the Medical Research Council.

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Authors and Affiliations

  1. School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K.

    Colin Kleanthous, Ulrike C. Kühlmann, Ansgar J. Pommer, Richard James & Andrew M. Hemmings

  2. School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, U.K.

    Neil Ferguson & Sheena E. Radford

  3. School of Chemical Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K.

    Geoffrey R. Moore & Andrew M. Hemmings

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  1. Colin Kleanthous
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Correspondence to Colin Kleanthous or Andrew M. Hemmings.

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Kleanthous, C., Kühlmann, U., Pommer, A. et al. Structural and mechanistic basis of immunity toward endonuclease colicins . Nat Struct Mol Biol 6, 243–252 (1999). https://doi.org/10.1038/6683

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