Molecular docking.

(A) Co-crystal ligand UDP can be well re-docked into its own binding pocket with RMSD = 0.353 Å. After the re-docking, UDP-GlcNAc was docked into the binding pocket with the co-crystal peptides present, performing the same conformation as UDP. Red stick: co-crystal UDP in 3PE4. Green surface: surface of hydrophobic residues. Blue surface: surface of hydrophilic residues and GLY. (B) There were four possible hydrogen-bond between GlcNAc and transferase; i.e. C6-OH∼Thr560, C4-OH∼Leu653, C3-OH∼Gly654 and the C2 N-acyl group∼His920. (C) The C2-acetamido points to a hydrophobic cave constituted by Met501, Leu502 and Tyr841. (D) There were six possible hydrogen-bonds between UDP-GlcNAc and O-GlcNAc transferase with a peptide substrate. Two of them were from UDP. One was between phosphate and Lys842. The other was between phosphate and Ser21 of the peptide substrate. The yellow dashed lines show the potential hydrogen-bonds. Green surface: surface of hydrophobic residues. Blue surface: surface of hydrophilic residues and GLY. Purple line: CKII peptide. The PyMOL molecular graphics system (Version 1.3r1 Schrödinger, LLC, USA) was used to conduct polarity analysis following manufacturer’s instruction [43].