Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD

Abstract

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP’s ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD’s antagonistic activities, despite a similar mode of engagement of its two substrates — unmodified and AMPylated BiP.

Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD

“…Solubilised His-FICD adopted a single oligomeric state as judged by SEC analysis ( Figure 5 B), as is the case for N-terminally truncated FICD, which forms a highly stable homodimer in the solution and in all published structures, e.g. [ 10 , 11 , 12 ]. To assess the oligomeric state of His-FICD experimentally, we employed native mass spectrometry, but were unfortunately not able to detect the protein by this method (Drs.…”

“…( A ) FICD is comprised of a short N-terminal cytosolic tail, a transmembrane domain (TM; dark grey), two tetratricopeptide repeats (TPR; turquoise), a linker (yellow), and the catalytic Fic domain (FIC; blue). The segment for which crystal structures have been solved (approximately residues 102 to 445 depending on the particular study) is indicated [ 10 , 11 , 12 , 22 ]. The numbers denote residue positions that mark the boundaries of key regions in the structure of FICD.…”

“…FICD catalyses two antagonistic reactions by utilising an oligomerisation-induced switch in the structure of the active site; AMPylation by the monomeric enzyme and deAMPylation by the dimeric enzyme [ 4 , 9 , 10 , 11 , 12 ]. These FICD-mediated reactions are important for the maintenance of ER proteostasis [ 4 , 5 , 13 , 14 ] and neuronal function [ 6 , 15 , 16 , 17 , 18 , 19 ], as well as adaptive immunity [ 19 ].…”

“…Moreover, while single-pass membrane proteins such as FICD represent roughly half of all membrane proteins, structural studies of these are limited [ 24 , 29 , 30 ]. The currently available crystal structures of FICD include the catalytic domain and the adjacent tetratricopeptide repeats (TPRs) ( Figure 1 B) [ 10 , 11 , 12 , 22 ]. In vitro investigations of FICD’s structure and enzymatic mechanism have been conducted using N-terminally truncated (lacking the first approximately 100 residues), soluble protein variants produced in E. coli .…”

A homozygous p.(Arg371Ser) mutation in FICD de-regulates AMPylation of the human endoplasmic reticulum chaperone BiP causing infancy-onset diabetes and severe neurodevelopmental delay

“…This biochemical feature is consistent with earlier observations that levels of modified (AMPylated) BiP are regulated physiologically: decreasing with mounting ER stress and increasing with stress resolution (Hendershot et al, 1988; Laitusis et al, 1999). This balancing act is critically-dependent on FICD (Casey et al, 2017; Preissler et al, 2017a) and arises from fine tuning of the enzyme’s active site to reciprocally regulate its two antagonistic functions (Perera et al, 2021; Perera et al, 2019).…”