University of Cambridge
Post-translational regulation of BiP by FICD-mediated AMPylation and deAMPylation
Abstract
dc:description.abstractRegulation of the amount and activity of Binding Immunoglobulin Protein (BiP) contributes to protein-folding homeostasis. BiP’s abundance is modulated transcriptionally by the canonical unfolded protein response (UPR). Conversely, a metazoan-specific, endoplasmic reticulum (ER)-resident, Fic domain containing protein (FICD) is able to dynamically adjust BiP’s activity through AMPylation and deAMPylation. These two mutually antagonistic reactions, catalysed by the single active site of FICD, are reciprocally regulated by an oligomeric state-dependent switch. Under conditions of low unfolded protein load this bifunctional (monomeric) Fic enzyme AMPylates and inactivates excess ATP-bound BiP. However, with increasing ER stress dimeric FICD rapidly deAMPylates the inactive BiP-AMP store — enabling extra BiP to re-enter the chaperone cycle and thereby increase the organelle’s chaperone capacity (in a post-translational strand of the UPR). In this thesis, through structural, biochemical and biophysical techniques, I address the fundamental nature of FICD’s post-translational regulation of BiP. By obtaining high-resolution crystal structures of trapped deAMPylation complexes (of FICD•BiP-AMP) I elucidate the basis of FICD substrate engagement, reveal the mechanism of Fic domain deAMPylation and clarify the essential role of the gatekeeper Glu234 residue (characteristic of the Fic domain inhibitory -helix) in this hydrolytic reaction. These structures also explain FICD’s exquisite selectivity for its AMPylation substrate — ATP-bound, domain-docked BiP — with FICD’s tetratricopeptide repeat domain binding a tripartite assembly of BiP’s nucleotide binding domain, docked linker and substrate binding domain, that is unique to the aforementioned Hsp70-state. My studies also shed light on the structural basis of the monomerisation-dependent switch between FICD’s two mutually antagonistic activities — which centres on a monomerisation-induced increase in gatekeeper Glu234 flexibility. Upon monomerisation, increased Glu234 flexibility permits AMPylation competent binding of MgATP in FICD’s active site whilst simultaneously impairing the ability for Glu234 to properly align an attacking water molecule for efficient deAMPylation of BiP-AMP.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2021
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Perera, Luke A
- Advisor dc:contributor.advisor
-
- Ron, David
Subjects
dc:subject × 16Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
- 0000-0002-3014-5636
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/319721