A proactive role of water molecules in acceptor recognition by protein O-fucosyltransferase 2

Abstract

Protein O-fucosyltransferase 2 (POFUT2) is an essential enzyme that fucosylates serine and threonine residues of folded thrombospondin type 1 repeats (TSRs). To date, the mechanism by which this enzyme recognizes very dissimilar TSRs has been unclear. By engineering a fusion protein, we report the crystal structure of Caenorhabditis elegans POFUT2 (CePOFUT2) in complex with GDP and human TSR1 that suggests an inverting mechanism for fucose transfer assisted by a catalytic base and shows that nearly half of the TSR1 is embraced by CePOFUT2. A small number of direct interactions and a large network of water molecules maintain the complex. Site-directed mutagenesis demonstrates that POFUT2 fucosylates threonine preferentially over serine and relies on folded TSRs containing the minimal consensus sequence C-X-X-S/T-C. Crystallographic and mutagenesis data, together with atomic-level simulations, uncover a binding mechanism by which POFUT2 promiscuously recognizes the structural fingerprint of poorly homologous TSRs through a dynamic network of water-mediated interactions.

Figure 1. Structure of CePOFUT2 in complex with GDP and HsTSR1

a, Cartoon representation of the complex. The N- and C-termini of CePOFUT2 are colored in pink and bluewhite, respectively. Secondary structures of HsTSR1 are colored in blue whereas loops and unstructured regions are in black. The disulfide bridges are indicated in yellow. The GlcNAc moiety covalently bound to N205 and GDP are shown in green carbon atoms while N205 is shown as pink carbon atoms. The acceptor S17 of HsTSR1 is shown as black carbon atoms. The flexible linker is shown in orange. b, Surface representation of the complex in two different views. c, Multiple sequence alignment of human TSR1, TSR2 and TSR3 of thrombospondin 1 and Rattus novergicus F-spondin 1 and 4 (upper panel). The fucosylated S/T residues and the conserved cysteines are indicated in red and yellow, respectively. The numbering for each TSR does not correspond to the numbering to their location in thrombospondin and F-spondin, respectively. Residues delimited by arrows are exposed to the bulk solvent. The consensus sequence, C¹X^a[X^b]X^c(S/T)C²X^dX^eG, is shown below the multiple sequence alignment. The lower panel shows a cartoon representation of HsTSR1 colored in black. The residues belonging to the CWR-layered structure and some other conserved residues (shown in bold) between human TSR1, TSR2 and TSR3 are shown as sticks with black carbon atoms. d, Diagram showing the different arrangement of disulfide bridges found in TSRs of group 1 and 2. The inverted triangles in red point out the fucosylation sites that in turn have the same location in the 3D fold of both groups. e, Surface representation of the CePOFUT2 (left) and HsTSR1 (right), colored by sequence conservation. GDP is shown as sticks with green carbon atoms.

Figure 2. Catalytic mechanism of POFUT2 and its preferences on threonine over serine residues

a, Close-up view of the complex active site. The residues of CePOFUT2 and the HsTSR1 are depicted as grey and orange carbon atoms, respectively. GDP is shown as green carbon atoms. Hydrogen bond interactions are shown as dotted green lines. Electron density maps are F_O–F_C and 2F_O–F_C syntheses (blue) contoured at 2.2 and 1.0 σ for GDP and S17/E52, respectively. b, Close-up view of CePOFUT2 in complex with GDP-fucose and HsTSR1. c, Relative fucosylation of HsTSR3 compared to the mutants T17S and T17A (Left panel). Close-up view of CePOFUT2 in complex with GDP-fucose and the mutant S17T of HsTSR1 (Right panel). To note that the hydroxyl group of S17 and T17 is 3.28 and 3.34 Å to the anomeric carbon of GDP-fucose, respectively. Both complexes with GDP-fucose were obtained by MD simulations (see Online methods). All replicates were in triplicate. Error bars are standard deviation, and p-values were calculated using ANOVA.* p<0.10, ** p<0.05, ***p<0.01

Figure 3. Interactions in the interface of the complex

a, Surface representation of the complex interface in two different views. Colors are the same as in Fig. 1b. Residues colored in grey and black are engaged in direct or water-mediated interactions. Water molecules are indicated as red spheres. b, Close-up view of the interface showing the only stacking interactions in the complex. c, Close-up view of the direct and water-mediated interactions present in the complex. CePOFUT2 and HsTSR1 are depicted as grey and orange, respectively. Residues carbon atoms for each protein are also shown with the same color above. d, Close-up view of the few direct interactions present in the complex. Colors are the same as above. Hydrogen bond interactions are shown as dotted green lines. e, Relative fucosylation of HsTSR3 in comparison with different mutants located in the interface. The activity for some mutants is compared with HsTSR3 either from lysates or media (see Supplementary Fig. 15). All replicates were in triplicate. Error bars are standard deviation, and p-values were calculated using ANOVA.* p<0.10, ** p<0.05, ***p<0.01

Figure 4. Hydration structure and dynamics of CePOFUT2-GDP-fucose-HsTSR1 supports water-mediated binding

a, Water oxygen density over 0.5 μs calculated for the ternary complex through MD simulations. The two domains of CePOFUT2-GDP are shown in grey and pink, respectively, HsTSR1 in blue and GDP-fucose as green sticks. Reactive S17 is highlighted in orange. Regions with water density greater than 1.5 times the density of the bulk are represented as blue isosurfaces. b, Examples of 1D-rdf functions calculated for buried (upper panel) and solvent-exposed (lower panel) atoms of HsTSR1 in the isolated (in orange) and bound (in blue) states. c, (Upper panel) 2D-rdf function calculated for two selected atoms of the protein-protein interface hydrogen bonded to a crystallographic water, together with its significance (population in the MD ensemble) and average residence time. The atoms are labeled according to AMBER force field nomenclature. (Lower panel) Ensembles for the ternary complex obtained through MD simulations for the residues involved in the water pocket. One conformation of the rest of the residues is shown for clarity.