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Molecular basis of Pirh2-mediated p53 ubiquitylation

Nature Structural & Molecular Biology volume 15, pages 1334–1342 (2008)Cite this article

Abstract

Pirh2 (p53-induced RING-H2 domain protein; also known as Rchy1) is an E3 ubiquitin ligase involved in a negative-feedback loop with p53. Using NMR spectroscopy, we show that Pirh2 is a unique cysteine-rich protein comprising three modular domains. The protein binds nine zinc ions using a variety of zinc coordination schemes, including a RING domain and a left-handed β-spiral in which three zinc ions align three consecutive small β-sheets in an interleaved fashion. We show that Pirh2-p53 interaction is dependent on the C-terminal zinc binding module of Pirh2, which binds to the tetramerization domain of p53. As a result, Pirh2 preferentially ubiquitylates the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 regulates protein turnover of the transcriptionally active form of p53.

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Figure 1: Sequence and domain organization of Pirh2.
Figure 2: Ribbon representations of the three individual Pirh2 domains and zinc coordination schemes of Pirh2.
Figure 3: Pirh2 interacts with p53 by a two-site binding mode.
Figure 4: Mapping UBE2D2 binding interface.
Figure 5: Pirh2-mediated ubiquitylation of itself and p53.
Figure 6: Ubiquitylation activity of Pirh2 deletion mutants.
Figure 7: Model of a potential ternary complex between Pirh2, p53 and the E2 enzyme UBE2D2.

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Acknowledgements

We thank A. Ayed for helpful comments on the manuscript and members of the Arrowsmith lab for technical advice and discussion. This work was funded by the Canadian Cancer Society through grants from the National Cancer Institute of Canada, the Protein Structure Initiative of the National Institutes of Health (P50-GM62413-01) through the Northeast Structural Genomics Consortium, and the Canada Research Chairs program (to C.H.A.). Y.S. is supported by a fellowship from the Leukemia and Lymphoma Research Society of Canada.

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Author notes

  1. Rob C Laister and Alexander Lemak: These authors contributed equally to this work.

Authors and Affiliations

  1. Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, M5G 1L7, Ontario, Canada

    Yi Sheng, Rob C Laister, Alexander Lemak, Bin Wu, Elizabeth Tai, Shili Duan, Jonathan Lukin, Sampath Srisailam, Murthy Karra & Cheryl H Arrowsmith

  2. Molecular Biotechnology, IFM, Campus Valla, Linköping University, S-581 83, Linköping, Sweden

    Maria Sunnerhagen

  3. Department of Biology, York University, Toronto, M3J 1P3, Ontario, Canada

    Yi Sheng & Sam Benchimol

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Contributions

Y.S., R.C.L. and E.T. designed and conducted experiments; Y.S., A.L., B.W. and M.S. determined the structures; R.C.L., J.L., S.S. and M.K. collected NMR data; S.D. provided constructs and reagents; and Y.S. and C.H.A. wrote the paper with input from all other authors.

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Correspondence to Cheryl H Arrowsmith.

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Sheng, Y., Laister, R., Lemak, A. et al. Molecular basis of Pirh2-mediated p53 ubiquitylation. Nat Struct Mol Biol 15, 1334–1342 (2008). https://doi.org/10.1038/nsmb.1521

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