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Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

Nature Chemical Biology volume 12, pages 735740 (2016) | Download Citation

Abstract

Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development, including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi's critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary and ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and a conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling–Degos disease are clustered around the enzyme active site and adversely affect its activity. Our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.

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Acknowledgements

We thank members of the Li and Haltiwanger labs for critical comments on this work, as well as S. Singh Johar for technical assistance. The work was supported by the NIH (grants GM061126 (to R.S.H.) and AG029979 (to H.L.)) and SBU–BNL (seed grant to R.S.H. and H.L.). We acknowledge access to beamlines X25, X29 and X4A at NSLS, Brookhaven National Laboratory and LRL-CAT at APS, Argonne National Laboratory, and we thank the staff at these beamlines. NSLS and APS were supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract nos. DE-AC02-98CH10886 and DE-AC02-06CH11357, respectively. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility. The results published here are in part based on data generated by the TCGA Research Network (http://cancergenome.nih.gov/). H.L. dedicates this work to the loving memory of his son Paul J. Li.

Author information

Author notes

    • Hongjun Yu
    •  & Hideyuki Takeuchi

    These authors contributed equally to this work.

Affiliations

  1. Biology Department, Brookhaven National Laboratory, Upton, New York, USA.

    • Hongjun Yu
    • , Qun Liu
    •  & Huilin Li
  2. Complex Carbohydrate Research Center, the University of Georgia, Athens, Georgia, USA.

    • Hideyuki Takeuchi
    • , Megumi Takeuchi
    •  & Robert S Haltiwanger
  3. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.

    • Joshua Kantharia
    •  & Huilin Li

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Contributions

H.Y., H.T., R.S.H. and H.L. designed the research. H.Y. prepared the protein complexes, performed crystallization and solved the structures. H.Y. and Q.L. collected and processed the sulfur-SAD data. H.Y. and H.L. analyzed the structures. H.T., M.T. and J.K. expressed and purified proteins and mutants and performed the enzymatic assays and mass spectrometry. H.Y., H.T., R.S.H. and H.L. designed the mutants, analyzed the mutant data and carried out the cancer-related analysis. H.Y. prepared the initial draft of the manuscript, and H.Y., H.T., R.S.H. and H.L. wrote the paper with input from all other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Robert S Haltiwanger or Huilin Li.

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    Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1–3.

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DOI

https://doi.org/10.1038/nchembio.2135

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