Fig. 6: The enzymatic mechanism of eukaryotic deAMPylation.

a BiP’s Thr518-AMP (purple) bound to FICD (yellow). Left, the arrangement of BiP’s AMPylated Thr518 and Mg²⁺ cation within the Fic domain active site. Residues interacting with Mg²⁺ and the AMP moiety are shown as sticks and annotated. Hydrogen-bonds involving the AMP moiety and FICD’s Glu234 sidechain are shown (with high confidence hydrogen bonds depicted with thick, pink dashed lines and those only meeting relaxed hydrogen bond constraints depicted with orange dashed lines). Note, the putative catalytic water molecule* forms hydrogen bonds to Glu234 (located at the top of α_inh) and potential hydrogen bonds to the backbone NH groups of the Fic domain anion-binding nest (G³⁶⁸NG³⁷⁰) and Arg371 (see Supplementary Fig. 8). Right, a slightly rotated view of the FICD-active site, shown on the left, overlaid with an unbiased polder OMIT electron density map, contoured at 4σ. For clarity only hydrogen bonds formed by Glu234 are shown (pink dashed lines). b As in the right-hand side of a but reduced to highlight Glu234’s coordination of the catalytic water molecule* and its position in-line for nucleophilic attack into the α-phosphate. Additionally, the putative general acid, His363, is modelled based on an alignment of FICD:MgATP (PDB 6I7K, turquoise)¹⁹. See Supplementary Movie 3.