Structural Insights into the Mechanism of Protein O-Fucosylation

Abstract

Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel SN1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

“…This may suggest that GalNAc-T11 is unique among the GalNAc-T isoforms in recognizing folded domains as substrates. Other glycosyltransferases initiating O-Fuc, O-Glc, and O-GlcNAc recognize small folded domains such as epidermal growth factor-like (EGF) and thrombospondin type 1 repeats (TSR) (26,36), and these domains share a design with three conserved disulphide bridges with the LA-modules (37)(38). However, in all cases the acceptor sites are located in the folded domain, while the substrate site for GalNAc-T11 is located in the short linker between the folded domains.…”

O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

“…This amino acid substitution is located in the GDP-fucosebinding motif and largely abolishes the O-fucosyltransferase activity in vitro (Fig. 1A) (23,55,56). We first sought to confirm that the O-fucosyltransferase activity of O-fut1 R245A knock-in was also negligible in vivo.…”

Novel roles for O-linked glycans in protein folding

“…The crystal structure of C. elegans Pofut1 showed the enzyme to have a classic GT-B fold formed by two Rossman domains [58]. GDP-fucose, the donor substrate in an O- fucosylation reaction, binds at the base of a large pocket nested between these two domains large enough to accommodate an EGF repeat (Figure 2A).…”

“…A) Pofut1 contains two Rossman-like folds with the GDP-fucose binding pocket nested deep between them [58]. The pocket is large enough to hold an EGF as well.…”