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A split active site couples cap recognition by Dcp2 to activation

Nature Structural & Molecular Biology volume 17, pages 1096–1101 (2010)Cite this article

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Abstract

Decapping by Dcp2 is an essential step in 5′-to-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step by two orders of magnitude and block the formation of the closed state, whereas Dcp1 enhances the catalytic step by a factor of 10 and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping where coactivators trigger decapping by stabilizing a labile composite active site.

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Figure 1: Mutants along the interdomain interface in the closed crystal form attenuate the catalytic step of decapping.
Figure 2: Some interface mutants block conversion to the closed form measured by SAXS.
Figure 3: The regulatory domain has a specific binding site for m7G.
Figure 4: The catalytic domain binds m7GDP on the catalytic helix and binds a variety of nucleotides along the RNA binding region.
Figure 5: Decapping by Dcp2 proceeds by the formation of a composite active site involving both the regulatory and catalytic domains.

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Acknowledgements

We thank M. Kelly for NMR support; K. Krukenberg as well as beamline scientists G. Hura and M. Hammel of Beamline 12.3.1 at the Advanced Light Source for assistance with SAXS experimentation and data analysis; and J.J. Miranda, M. Pufall, G. Narlikar and D. Morgan for critical reading of the manuscript. This work was supported by US National Institutes of Health grant R01GM078360 to J.D.G. S.N.F. received support from the Sandler Family Foundation for Basic Sciences and the Achievement Awards for College Scientists Foundation. B.N.J. was supported by a National Science Foundation predoctoral fellowship. G.A.H. was a student in the University of California, San Francisco Summer Research Training Program. The Advanced Light Source is supported by US Department of Energy Contract No. DE-AC02-05CH11231.

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  1. Gail A Hernandez

    Present address: Present address: Department of Chemistry, University of Chicago, Chicago, Illinois, USA.,

Authors and Affiliations

  1. Graduate Group in Biophysics, University of California, San Francisco, California, USA

    Stephen N Floor

  2. Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA

    Stephen N Floor, Brittnee N Jones, Gail A Hernandez & John D Gross

  3. Program in Chemistry and Chemical Biology, University of California, San Francisco, California, USA

    Brittnee N Jones

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Contributions

S.N.F. designed and performed experiments, analyzed data, wrote the manuscript and prepared the figures; B.N.J. performed an experiment; G.A.H. generated Dcp2 mutants; J.D.G. supervised the project and experimental design and guided manuscript preparation.

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Correspondence to John D Gross.

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Floor, S., Jones, B., Hernandez, G. et al. A split active site couples cap recognition by Dcp2 to activation. Nat Struct Mol Biol 17, 1096–1101 (2010). https://doi.org/10.1038/nsmb.1879

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