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Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism

Abstract

A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α-1,3-xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor–like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.

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Figure 1: The mouse XXYLT1 is a dimer and has a GT-A fold with its active site facing sideways to facilitate lateral modification of Notch.
Figure 2: The Xyl-Glc-EGF acceptor substrate undergoes a large conformational change when bound to XXYLT1.
Figure 3: Snapshots of XXYLT1 along its retaining reaction pathway.
Figure 4: Proposed retaining mechanism of XXYLT1.
Figure 5: Notch signaling in certain cancers may be inhibited by alterations in XXYLT1.

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Acknowledgements

We thank members of the Li and Haltiwanger labs for critical comments on this work. The work was supported by US National Institutes of Health grants GM061126 (R.S.H.) and AG029979 (H.L.), Stony Brook University–Brookhaven National Laboratory Seed grant (R.S.H. and H.L.), German Research Foundation (Deutsche Forschungsgemeinschaft (DFG)) grant BA4091/5-1 (H.B.) and National Science Foundation grant DMR 1404985 (E.L.). We acknowledge access to beamlines X25, X29 and X6A at the National Synchrotron Light Source (NSLS) and thank the staff at these beamlines. NSLS were supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-98CH10886. UDP-xylose isolation by the Carbosource Services at the Complex Carbohydrate Research Center, University of Georgia was supported in part by the US Department of Energy grant DE-FG02-93ER20097. The results published here are in part based upon data generated by the Cancer Genome Atlas Research Network (http://cancergenome.nih.gov/).

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Contributions

H.Y., H.B., R.S.H., H.L. and H.T. designed the research. H.Y. prepared the complexes, performed crystallization and solved the structures. H.Y. and H.L. analyzed the structures. M.T. and H.T. expressed and purified proteins and mutants, and performed the enzymatic assays. H.Y., R.S.H., H.L. and H.T. designed the mutants, analyzed the mutant data, performed the cancer-related analysis and wrote the paper. J.L., J.K. and E.L. joined this project after the original submission of the manuscript and contributed to the biophysical experiments regarding the folding status of wild-type and mutated XXYLT1 proteins, which was essential for the revisions to the manuscript.

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Correspondence to Huilin Li or Hideyuki Takeuchi.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–11 and Supplementary Tables 1–4. (PDF 9008 kb)

A movie morphing the isolated hFA9 EGF (PDB 1EDM) to the same protein in complex with XXYLT1 (MOV 19050 kb)

41589_2015_BFnchembio1927_MOESM292_ESM.mov

Morphing (generated using Chimera) of the electron densities of donor and acceptor substrates of the four trapped ternary complexes. (MOV 13771 kb)

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Yu, H., Takeuchi, M., LeBarron, J. et al. Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism. Nat Chem Biol 11, 847–854 (2015). https://doi.org/10.1038/nchembio.1927

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