An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP

Abstract

AMPylation is an inactivating modification that matches the activity of the major endoplasmic reticulum (ER) chaperone BiP to 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 in residues tracing an inhibitory relay from 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. Whereas, 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.Impact StatementUnique amongst known chaperones, the endoplasmic reticulum (ER)-localized Hsp70, BiP, is subject to transient inactivation under conditions of low ER stress by reversible, covalent modification – AMPylation. The enzyme responsible for this modification, FICD, is in fact a bifunctional enzyme with a single active site capable of both AMPylation and deAMPylation. Here we elucidate, by biochemical, biophysical and structural means, the mechanism by which this enzyme is able to switch enzymatic modality: by regulation of its oligomeric state. The oligomeric state-dependent reciprocal regulation of FICD activity is, in turn, sensitive to the ATP/ADP ratio. This allosteric pathway potentially facilitates the sensing of unfolded protein load in the ER and permits the transduction of this signal into a post-translational buffering of ER chaperone activity.

Post-translational modifications of Hsp70 family proteins: Expanding the chaperone code

“…These sites play slightly different roles in regulation of the Hsp70 ATPase cycle. Whereas AMPylation of Thr-366 increases basal ATPase activity with no effect on J-proteinstimulated ATPase activity, AMPylation of Thr-518 decreases J-protein-stimulated ATPase activity with no effect on basal ATPase activity (94,107). In addition to BiP, human FICD also AMPylates other major chaperones, including Hsp70, Hsp40, and Hsp90 (101).…”

“…AMPylation is considered to inhibit the activity of Hsp70 family chaperones, conserving a pool of chaperones in a "stand-by," or "primed," state, which can rapidly respond to stress when de-AMPylated (92,93). The AMPylase FICD functions as a bidirectional enzyme, catalyzing both AMPylation and de-AMPylation involving a single active site (93,94). This bidirectionality is regulated by the oligomeric state of FICD, with monomeric FICD acting as an AMPylator and the dimeric form de-AMPylating (92,95).…”

Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes

“…BiP for oligomer crystallisation Chinese hamster BiP T229A-V461F (residues 28-549) was expressed as an N-terminal His6-Smt3tagged protein (UK2090). The purification protocol was the same as used for His6-Smt3-FICD purification described in (Perera et al, 2019) with minor modifications. The protein was expressed in M15 E. coli cells grown in LB medium supplemented with 100 μg/ml ampicillin and 50 μg/ml kanamycin.…”

“…After anion exchange chromatography the protein was exchanged into 10 mM HEPES-KOH pH 7.4, 150 mM KCl, 2 mM MgCl2, and 1 mM TCEP by gel filtration and concentrated to 21 mg/ml. For crystallisation, BiP and an equimolar amount of human FICD L258D-H363A [residues 104-445; UK2093, purified as in (Perera et al, 2019)] were diluted to 150 µM with TNKM (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 50 mM KCl and 2 mM MgCl2) and supplemented with 175 µM ATP. Note: FICD was excluded from the resulting crystals of oligomeric BiP (see below).…”

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, ). The endoplasmic reticulum protein huntingtin yeast‐interacting 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, ; Figure ). This involves an allosteric‐based AMPylation‐competent recruitment of Mg‐ATP.…”