Abstract
Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle.
Highlights
Protein O-fucosyltransferase 1 (PoFUT1) is a glycosyltransferase (GT) present in the endoplasmic reticulum that catalyzes the transfer of a fucose (Fuc) residue from GDP-Fuc directly into Ser/Thr residues of some proteins containing EGF-like repeats [1,2]
The dimers present a certain overlap of one of the domains that, together with the structural reorganization, makes the overall height above the plane twice that observed by the rest of the monomeric species (Figure 3a). We studied this dimerization in solution at the singlemolecule level using the singly-labeled constructs, CePoFUT1-ATTO 488 (A488) and CePoFUT1-ATTO 647N (A647N), mixed at stoichiometric ratio
These GTs display the closure of the active site upon donor sugar nucleotide binding, which appears a prior step for enzyme catalysis [19]
Summary
PoFUT1 is a glycosyltransferase (GT) present in the endoplasmic reticulum that catalyzes the transfer of a fucose (Fuc) residue from GDP-Fuc directly into Ser/Thr residues of some proteins containing EGF-like repeats [1,2]. These receptors are transmembrane proteins that are the core of the Notch signaling pathway They can have ~29–36 EGF like-repeats [4], that can be fucosylated by. The importance of the addition of a Fuc residue into Notch receptors has been deeply studied in the past years, e.g., a Fuc moiety promotes the interaction between Notch and its ligands [8,9]. This was revealed by the recent crystallographic data that demonstrated how a Fuc residue located at Notch EGF12 contributes significantly to the contact with delta like ligand 4 (DLL4) [8] and Jagged1 [9]. The Fuc moiety is further elongated with a N-acetylglucosamine (GlcNAc) residue by the Fringe
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