Regulation of Notch signaling by O‐glucosylation: Notch‐modifying xylosyltransferase‐substrate complexes 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. Importantly, XXYLT1 is amplified in a number of squamous cell carcinomas and may be a target for therapeutics. 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.Support or Funding InformationThe work is supported by NIH grants GM061126 (to R.S.H) and AG029979 (to H.L.), SBU‐BNL Seed Grant (to R.S.H. and H.L.), DFG grant BA4091/5‐1 (to H.B.) and NSF grant DMR 1404985 (to E.L.).