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

Abstract

A major remaining question in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xylosideα1–3 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 where dysregulation of Notch is known to cause cancer or developmental disorders.

Does Notch play a tumor suppressor role across diverse squamous cell carcinomas?

“…A nice example is a recent research on the tumor suppressor role of Notch pathway in bladder cancer, revealed by the characterization of multiple components of Notch pathway 100. We have recently characterized a Notch‐modifying enzyme Xxylt1 101 that is frequently amplified in specific cancer types lacking loss‐of‐function XXYLT1 mutations. Surprisingly, of the six cancer types with highest XXYLT1 amplification frequency, three are squamous cell carcinomas: SCCs of head and neck, lung, and cervical.…”

The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force

“…Yun et al solved X-ray structures of the endoplasmic reticulum enzyme Xyloside α-1,3-xylosyltransferase (XXYLT1), a well conserved type II membrane protein that can extend O-glucose modifications within Notch EGF repeats. In addition they solved the structure of Rumi alone and in complex with an EGF repeat derived from the coagulation factor IX containing a Xyl-Glc disaccharide modification (Yu et al, 2016; Yu et al, 2015). These structures provided mechanistic insights into how these enzymes bind to and modify Notch.…”

“…These structures provided mechanistic insights into how these enzymes bind to and modify Notch. In the apo structure (the enzyme without a substrate), XXYLT1 forms a dimer such that the enzyme active sites are in an ideal conformation to interact with laterally oriented Notch EGF repeats within ER lumen (Yu et al, 2015). The structure of XXYLT1 is largely unchanged when in complex with the EGF repeat but unexpectedly, the structure of the EGF repeat undergoes a large conformational change while maintaining all disulfide bonds.…”

Recent structural and mechanistic insights into protein O-GalNAc glycosylation

“…In the presence of such a group, a double-displacement mechanism would ensue with the intermediacy of a covalent glycosyl-enzyme adduct [1,7]. Whereas, in the absence of such a putative nucleophile, a front-face reaction pathway has been postulated with the formation of a short-lived oxocarbenium ion-like intermediate [8][9][10][11][12] (Figure 1a). …”