Recognition of EGF-like domains by the Notch-modifying O-fucosyltransferase POFUT1

Abstract

Protein O-fucosyltransferase 1 (POFUT1) fucosylates the epidermal growth factor (EGF)-like domains found in cell-surface and secreted glycoproteins including Notch and its ligands. Although Notch fucosylation is critical for development, and POFUT1 deficiency leads to human disease, how this enzyme binds and catalyzes the fucosylation of its diverse EGF-like domain substrates has not been determined. Reported here is the X-ray crystal structure of mouse POFUT1 in complex with several EGF-like domains, including EGF12 and EGF26 of Notch. Overall shape complementarity, interactions with invariant atoms of the fucosylation motif and flexible segments on POFUT1 all define its EGF-like-domain binding properties. Using large-scale structural and sequence analysis, we also show that POFUT1 binds EGF-like domains of the hEGF type and that the highly correlated presence of POFUT1 and fucosylatable hEGFs has accompanied animal evolution.

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Figure 1: Overall structure of the POFUT1–EGF-LD complexes.
Figure 2: The conserved EGF-LD interactions with POFUT1.
Figure 3: The EGF-LD O-fucosylation motif and the catalytic site of POFUT1.
Figure 4: POFUT1 interactions with the C1–C2 loop and the C5–C6 subdomain of the EGF-LDs.
Figure 5: The average structural maps (ASMs) of the hEGF, cEGF, lamEGF and intEGF domains.

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Acknowledgements

This work was supported by a grant to J.M.R. from the Canadian Institutes of Health Research, Funding Reference Number MOP-125956. X-ray crystallographic data were collected at beamline 08ID-1 at the Canadian Light Source. We also thank the Canadian Macromolecular Crystallography Facility staff for the mail-in data that were collected.

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Z.L., J.E.P. and J.M.R. designed the research. Z.L., K.H., J.E.P. and M.S. performed the experiments. Z.L. carried out the bioinformatic and structural studies. M.S. and D.Z. provided technical assistance. Z.L. and J.M.R. wrote the manuscript.

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Correspondence to James M Rini.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–8 and Supplementary Figures 1–14 (PDF 13616 kb)

Supplementary Dataset 1

Statistics for the distribution of EGF-like domain (EGF-LD) types and Ofucosylation motifs in 339 animal species (PDF 7901 kb)

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Li, Z., Han, K., Pak, J. et al. Recognition of EGF-like domains by the Notch-modifying O-fucosyltransferase POFUT1. Nat Chem Biol 13, 757–763 (2017). https://doi.org/10.1038/nchembio.2381

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