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

Abstract

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER‐localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation‐competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide‐binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.

Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria

“…Some Fic proteins such as dFic from Drosophila , HYPE, and EfFIC from Enterococcus faecalis harbor an intramolecular inhibitory motif and their activity is influenced by the oligomeric state of the enzyme and the presence of certain metal ions ( Casey et al, 2017 ; Preissler et al, 2017 ; Perera et al, 2019 ; Veyron et al, 2019 ). Similar to these proteins, Fic-2 harbors an inhibitory motif in its amino terminal portion capable of blocking its AMPylation activity.…”

“…AMPylation induced by Fic proteins or other AMPylators is reversible by specific enzymes ( Tan and Luo, 2011 ). Recent interesting studies reveal that the human Fic protein HYPE displays a deAMPylase activity in response to fluctuations in the unfolded protein load ( Casey et al, 2017 ; Preissler et al, 2017 ; Perera et al, 2019 ; Veyron et al, 2019 ). Whether and how the activity of Fic proteins from other organisms is regulated awaits further investigation.…”

Fic and non-Fic AMPylases: protein AMPylation in metazoans

“…While some kinetic studies point to a sequential reaction where auto-AMPylation is followed by target AMPylation, other studies hypothesize a substrate-assisted attack (figure 6), where the conserved His functions as a proton acceptor (accepts a proton from a substrate Thr/Tyr), resulting in a subsequent nucleophilic attack by these residues on the α-phosphate of ATP [35]. WT HYPE acts primarily as a deAMPylase [77,84,85] and the deAMPylation mechanism proposed by David Ron's group [77] suggests that Glu234 is actively engaged (the positively charged Arg374 holds the negatively charged Glu234 side chain in proximity to the active site) in coordinating a water molecule which acts as a nucleophile and attacks the phosphodiester bond between the hydroxyl group of a target residue (BiP Thr518) and the AMP moiety attached to the active site of HYPE (figure 6). Consequently, the AMP moiety is removed from the catalytic site and His363, which in the AMPylation reaction deprotonates and hence, primes the hydroxyl group of Thr518 to act as a nucleophile, now protonates the leaving Thr518 group [77].…”

Cellular microbiology: Bacterial toxin interference drives understanding of eukaryotic cell function

“…The discovery that AMPylase bacterial effectors share a conserved FIC domain led to the subsequent discovery that AMPylation is shared by eukaryotic cells (Veyron, Peyroche, & Cherfils, 2018). The endoplasmic reticulum protein huntingtin yeastinteracting protein E regulates the unfolded protein response in cells through AMPylation (monomeric form) and de-AMPylation (dimeric form) of the binding immunoglobulin protein molecular chaperone, which is also a target of the subtilase cytotoxin from several pathogenic strains of E. coli (Perera et al, 2019; Figure 1). This involves an allosteric-based AMPylation-competent recruitment of Mg-ATP.…”