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

Nat Chem Biol. 2015 Nov;11(11):847-54. doi: 10.1038/nchembio.1927. Epub 2015 Sep 28.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biocatalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • Endoplasmic Reticulum / chemistry
  • Endoplasmic Reticulum / enzymology
  • Epidermal Growth Factor / chemistry*
  • Epidermal Growth Factor / genetics
  • Epidermal Growth Factor / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Glycosylation
  • HEK293 Cells
  • Humans
  • Intracellular Membranes / chemistry
  • Intracellular Membranes / enzymology
  • Mice
  • Models, Molecular
  • Pentosyltransferases / chemistry*
  • Pentosyltransferases / genetics
  • Pentosyltransferases / metabolism
  • Protein Multimerization
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Receptor, Notch1 / chemistry*
  • Receptor, Notch1 / genetics
  • Receptor, Notch1 / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • UDP Xylose-Protein Xylosyltransferase
  • Xylose / chemistry*
  • Xylose / metabolism

Substances

  • Notch1 protein, mouse
  • Receptor, Notch1
  • Recombinant Proteins
  • Epidermal Growth Factor
  • Xylose
  • Pentosyltransferases

Associated data

  • PDB/4WLM
  • PDB/4WM0
  • PDB/4WMA
  • PDB/4WMB
  • PDB/4WMI
  • PDB/4WMK
  • PDB/4WN2
  • PDB/4WNH