The Essential Role of Water Molecules in the Reaction Mechanism of Protein O-Fucosyltransferase 2

Abstract

Protein O-fucosyltransferase 2 (PoFUT2) is an inverting glycosyltransferase (GT) that fucosylates thrombospondin repeats (TSRs) from group 1 and 2. PoFUT2 recognizes a large and diverse number of TSRs through a dynamic network of water-mediated interactions. By X-ray structural studies of C. elegans PoFUT2 complexed to a TSR of group 2, we demonstrate that this GT recognizes similarly the 3D structure of TSRs from both groups 1 and 2. Its active site is highly exposed to the solvent, suggesting that water molecules might also play an essential role in the fucosylation mechanism. We applied QM/MM methods using human PoFUT2 as a model, and found that HsPoFUT2 follows a classical S_N 2 reaction mechanism in which water molecules contribute to a great extent in facilitating the release of the leaving pyrophosphate unit, causing the H transfer from the acceptor nucleophile (Thr/Ser) to the catalytic base, which is the last event in the reaction. This demonstrates the importance of water molecules not only in recognition of the ligands but also in catalysis.

Keywords: Enzyme Catalysis; Fucosylation; Glycosyltransferases; Protein Glycosylation; QM/MM.

Figure 1

a) Multiple sequence alignment of HsTSR1, HsTSR2 and HsTSR3 of thrombospondin 1 and RnTSR1 and RnTSR4 of F‐spondin. Red arrowhead indicates fucosylated serine and threonine residues, and conserved Cys residues are highlighted in yellow. The numbering for each TSR does not correspond to its location in thrombospondin 1 or F‐spondin.

Figure 2

a) Cartoon representation of the complex. CePoFUT2^A418C and RnTSR4^E10C are shown in green and cyan, respectively. Disulfide bridges are indicated in yellow. Carbon atoms of the GlcNAc moiety covalently bound to Asn205 are shown in pink. A sulfate molecule and the acceptor Thr17 of RnTSR4^E10C are also shown in the active site. b) Close‐up view of the few direct interactions in the complex. H‐bond interactions are shown as dotted black lines.

Figure 3

a) Superimposed and aligned structures of the initial points (MD) corresponding to PoFUT2 (green) in complex with GDP‐fucose and RnTSR4 (cyan) and PoFUT2 (gray) in complex with GDP‐fucose and HsTSR1 (magenta). GDP‐fucose is showed in red at the active site exposed to solvent (red arrow). b), c) Detail of residues of PoFUT2 interacting with GDP‐fucose in the complex with HsTSR1 (b) and RnTSR4 (c). The Connolly surface for GDP‐fucose reflects the high accessibility (red) of the solvent to the pyrophosphate unit in both cases. d) Snapshot of MD simulations corresponding to the binary complex between HsPoFUT2 (green) and GDP‐fucose (gray). The reactive face of fucose anomeric carbon is oriented towards the opposite face of the catalytic base. e) Snapshot of MD simulations corresponding to the ternary complex between HsPoFUT2 (cyan), GDP‐fucose (gray) and HsTSR1 (magenta) The reactive face of fucose anomeric carbon has rotated to be oriented towards the same side in which Ser17 (the nucleophile) and Glu54 (the catalytic base) are placed. Both snapshots (d) and (e) have the same orientation view.

Figure 4

a) Detail of water molecules considered in the QM region, illustrated for the reagent corresponding to the glycosylation of HsTSR1. b) Potential energy surfaces for the different QM models tested with HsTSR1. QM1 corresponds to the model without waters (82 atoms). Successive models, QM2‐QM6, correspond to further incorporation of water molecules to the QM system as indicated. The model in QM6 (103 atoms), containing seven water molecules, showed the best result.

Figure 5

a) Transition structure TS1 corresponding to the glycosylation of HsTSR1 (cyan). b) Transition structure TS2 corresponding to the glycosylation of RnTSR4 (magenta). c) Overlay of the QM regions of the transition structures TS1 and TS2. TS1 and TS2 corresponding to the glycosylation of RnTSR4 (green, nucleophilic Thr17 in cyan) and HsTSR1 (gray, nucleophilic Ser17 in magenta), respectively. Note the alignment of the breaking/forming bonds (dashed black line). Pyrophosphate of TS2 is displayed in blue to distinguish the different orientation allowed by exposure to the solvent (the leaving oxygen is, however, perfectly aligned). QM regions are given in sticks.