A Ca²⁺-regulated deAMPylation switch in human and bacterial FIC proteins

Abstract

words) 21Significance statement (120 words) 44 45 Many FIC proteins regulate target proteins by addition of AMP, a reaction coined 46AMPylation. In a large FIC proteins subgroup, AMPylation is autoinhibited by a conserved 47 57 58In less than a decade, FIC proteins have emerged as a large family of enzymes controling the 59 activity of target proteins by post-translationally modifying them with phosphate-containing 60 compounds (reviewed in (1, 2 , 3 , 4 )). These proteins are characterized by the presence of a 61 conserved FIC domain, which carries out the post-translational modification (PTM) of a Tyr, 62Ser or Thr residue in a target protein (5 , 6 , 7 , 8 , 9 , 10 , 11). The most frequent PTM 63 reaction catalyzed by FIC enzymes is the addition of AMP using ATP as a cofactor, coined 64AMPylation or adenylylation. This PTM activity was originally discovered in toxins from 65 bacterial intracellular pathogens (12). It was later identified in bacterial toxin/antitoxins (e.g. 66 (7 )) and other bacterial FIC proteins of unknown functions (e.g. (11)), and in the only FIC 67 protein found in metazoans, HYPE/FicD, which controls the reversible AMPylation of the 68 BIP chaperone in the endoplasmic reticulum (ER) to match its activity to the load in unfolded 69 proteins (13 , 14 , 15). A commonality of AMPylation and all other PTM reactions catalyzed 70 by FIC proteins is that they use a motif of conserved sequence motif for catalysis, the FIC 71 motif, which carries an invariant histidine that is critical for nucleophilic attack of the cofactor 72 by the target residue, and an acidic residue (aspartate or glutamate) that binds an Mg 2+ ion to 73 stabilize the negative charges of the cofactor phosphates at the transition state (reviewed in (1, 74 2 , 3 , 4 )). 75 76Given their role in controling important bacterial and cellular responses, FIC-dependent PTM 77 levels are expected to be precisely regulated. For instance, FIC toxin components of toxin-78 antitoxin modules are inhibited by obstruction of their active sites by their cognate antitoxin, 79 and this strong inhibition is relieved by removal of the antitoxin (7 , 9 , 16). In a different 80 strategy, addition of phosphocholine to cellular GTPases by Legionella pneumophila AnkX is 81 reversed by another toxin, Lem3 (reviewed in (3)). Departing from these intermolecular 82 mechanisms, an intriguing autoregulatory glutamate has been described in various 83AMPylating FIC proteins, which protrudes into the catalytic site from either N-terminal 84 elements, as in human FicD (10), Clostridum difficile FIC (11) or Shewanella oneidensis FIC 85 (17), or from a C-terminal α-helix as in single-domain FIC proteins from Neisseria 86 meningitidis (7) and Helicobacter pylori (PDB 2F6S). This glutamate superimposes with an 87 inhibitory glutamate from the VbhA antitoxin that blocks the ATP-binding site in its cognate 88VbhT toxin (7), and its mutation into Ala or Gly has been consistently shown to increase 22). We discover that EfFIC has both AMPylation and deAMPylation activiti...

An oligomeric state‐dependent switch in the ER enzyme FICD regulates AMP ylation and de AMP ylation of BiP

“…Moreover, in vitro modification of BiP by purified FICD requires mutation of Glu234, suggesting that an AMPylation repressed state is favoured by wild‐type FICD. Remarkably, the Fic domain of FICD is also responsible for BiP deAMPylation: an activity that depends on Glu234 (Casey et al , ; Preissler et al , ) and magnesium (Veyron et al , ). These findings point to deAMPylation as the default activity of the bifunctional enzyme and implicate Glu234 in a functional switch between the two antagonistic activities of the Fic active site.…”

Fic and non-Fic AMPylases: protein AMPylation in metazoans

“…Increasing this ratio or removing Mg 2+ completely from the reaction mixture decreases the deAMPylation efficiency of WT HYPE. Conversely, WT HYPE efficiently catalysed BiP deAMPylation in the presence of only Mg 2+ [47]. This observation, coupled with similar results observed for the bifunctional Enterococcus faecalis Fic (EfFic), led the authors to propose a divalent metal ion differential as an alternative (or additional) mode of regulation for the displacement of Glu234 (inherent conformational flexibility) [47].…”

“…Together, this data suggests that monomeric HYPE mutants possess enhanced AMPylation function, and a slightly but significantly repressed deAMPylase function [85]. Recent work by Veyron et al has shown that the WT HYPE-mediated BiP deAMPylation can be fine-tuned by modulating the Ca 2+ /Mg 2+ ratio in vitro [47]. Increasing this ratio or removing Mg 2+ completely from the reaction mixture decreases the deAMPylation efficiency of WT HYPE.…”

“…Conversely, WT HYPE efficiently catalysed BiP deAMPylation in the presence of only Mg 2+ [47]. This observation, coupled with similar results observed for the bifunctional Enterococcus faecalis Fic (EfFic), led the authors to propose a divalent metal ion differential as an alternative (or additional) mode of regulation for the displacement of Glu234 (inherent conformational flexibility) [47]. These results raise the possibility that the FIC domain may act as an enzymatic calcium sensor, and prompt us to ask whether HYPE can respond to diffusible signals, such as a drop in ER calcium levels under stress conditions.…”

From Young to Old: AMPylation Hits the Brain

“…Even though the disruption of dimerization by an I271D mutation led to increased AMPylation activity in vitro, the deAMPylation activity in vivo was retained. Next, in vivo studies showed that human FICD deAMPylation activity was fully inhibited by the presence of Ca 2+ ions (Veyron et al, 2019). In comparison, Mg 2+ ions had no impact on FICD deAMPylation catalysis.…”