Structural and biochemical analysis of ATF6 and PERK: towards understanding the mechanism of UPR signal activation
File(s)
Author(s)
Adams, Christopher J.
Type
Thesis
Abstract
Within the endoplasmic reticulum (ER) of higher eukaryotes, the unfolded protein response (UPR) plays an essential role in restoring protein homeostasis during ER stress. This pathway is mediated by three core proteins, PERK, IRE1 and ATF6. The ER chaperone, BiP, has been suggested to act as an ER stress sensor by dissociating from the luminal domains of these UPR sensors upon binding to misfolded proteins, leading to UPR activation. Perturbation of this mechanism has been implicated in an array of cancers and neurodegenerative diseases, however, due to the experimental challenges in ATF6 protein production, structural and mechanistic understanding of ATF6 is lacking. Therefore, this thesis aimed to optimise the synthesis of ATF6 luminal domain (ATF6LD) and to investigate the hypothesis that it shares the same mechanism of activation observed for PERK and IRE1. Furthermore, structural insights into ATF6LD and PERK full-length (PERKFL) were pursued using electron microscopy (EM) to better understand their biological function and facilitate therapeutic strategies.
Extensive optimisation resulted in the successful expression and purification of native ATF6LD that was shown to adopt two distinct glycoform populations in the ER. In vitro biophysical studies identified a high affinity (Kd = 0.13 0.02 M) interaction between ATF6LD and the nucleotide binding domain (NDB) of BiP, indicating a shared non-canonical mode of binding, as previously proposed for PERK and IRE1. Low resolution EM analysis revealed the first structural insights of ATF6LD and showed that in the absence of BiP, ATF6LD can form a ring-like homo-trimer/tetramer complex that may play an important functional role.
The novel synthesis of soluble PERK enabled interaction studies which showed binding of PERKFL and a megacomplex, consisting of the 80S ribosome and translocon members. Cryo-EM structural analysis of the 80S ribosome and a suspected PERK density at the P-stalk was determined at 2.9 Å. Occupancy of this density was questioned at high resolution and the exact binding site of PERK remains to be determined. Data analysis also identified dynamics of the 40S subunit between POST translation and idle state ribosome populations revealing a pincer-like movement that blocks the ribosome E-site.
The results of this thesis reveal novel insights into the biological function of ATF6 and PERK. Developments in ATF6LD production and EM analysis will pave the way for further high resolution structure determination. The discovery that ATF6LD binds to the NBD of BiP significantly adds to our understanding of its mechanism of activation and provides an opportunity for designing new therapies. Binding of PERKFL to a translocon megacomplex suggests a link between the UPR and co-translational machinery.
Extensive optimisation resulted in the successful expression and purification of native ATF6LD that was shown to adopt two distinct glycoform populations in the ER. In vitro biophysical studies identified a high affinity (Kd = 0.13 0.02 M) interaction between ATF6LD and the nucleotide binding domain (NDB) of BiP, indicating a shared non-canonical mode of binding, as previously proposed for PERK and IRE1. Low resolution EM analysis revealed the first structural insights of ATF6LD and showed that in the absence of BiP, ATF6LD can form a ring-like homo-trimer/tetramer complex that may play an important functional role.
The novel synthesis of soluble PERK enabled interaction studies which showed binding of PERKFL and a megacomplex, consisting of the 80S ribosome and translocon members. Cryo-EM structural analysis of the 80S ribosome and a suspected PERK density at the P-stalk was determined at 2.9 Å. Occupancy of this density was questioned at high resolution and the exact binding site of PERK remains to be determined. Data analysis also identified dynamics of the 40S subunit between POST translation and idle state ribosome populations revealing a pincer-like movement that blocks the ribosome E-site.
The results of this thesis reveal novel insights into the biological function of ATF6 and PERK. Developments in ATF6LD production and EM analysis will pave the way for further high resolution structure determination. The discovery that ATF6LD binds to the NBD of BiP significantly adds to our understanding of its mechanism of activation and provides an opportunity for designing new therapies. Binding of PERKFL to a translocon megacomplex suggests a link between the UPR and co-translational machinery.
Version
Open Access
Date Issued
2020-11
Date Awarded
2021-05
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Ali, Maruf
Sponsor
Cancer Research UK
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)