Allosteric fine-tuning of the conformational equilibrium poises the chaperone BiP for post-translational regulation.

Lukasz Wieteska,Saeid Shahidi,Anastasia Zhuravleva

doi:10.7554/elife.29430

Abstract

BiP is the only Hsp70 chaperone in the endoplasmic reticulum (ER) and similar to other Hsp70s, its activity relies on nucleotide- and substrate-controllable docking and undocking of its nucleotide-binding domain (NBD) and substrate-binding domain (SBD). However, little is known of specific features of the BiP conformational landscape that tune BiP to its unique tasks and the ER environment. We present methyl NMR analysis of the BiP chaperone cycle that reveals surprising conformational heterogeneity of ATP-bound BiP that distinguishes BiP from its bacterial homologue DnaK. This unusual poise enables gradual post-translational regulation of the BiP chaperone cycle and its chaperone activity by subtle local perturbations at SBD allosteric 'hotspots'. In particular, BiP inactivation by AMPylation of its SBD does not disturb Hsp70 inter-domain allostery and preserves BiP structure. Instead it relies on a redistribution of the BiP conformational ensemble and stabilization the domain-docked conformation in presence of ADP and ATP.

Highlights

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells responsible for folding and maturation of the majority of secreted and membrane proteins
All of the experiments were performed using a well-established variant of wild-type (WT) binding protein (BiP), in which Thr 229 was mutated to Gly to slow down protein ATPase activity (Wei et al, 1995)
To monitor BiP conformational changes in the presence of different ligands and distinguish long-range conformational perturbations from local effects of ligand binding, we used the ‘divide-and-conquer’ approach (Ruschak and Kay, 2010; Gelis et al, 2007) that we had previously adopted for characterization of the E. coli BiP homolog DnaK (Zhuravleva et al, 2012)

Summary

Introduction

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells responsible for folding and maturation of the majority of secreted and membrane proteins. An immunoglobulin heavy-chain binding protein (BiP; known as GRP78 and HSP5A) is the only Hsp chaperone in the ER. BiP binds to the majority of unfolded and misfolded proteins in this organelle to promote their folding and prevent aggregation (Araki and Nagata, 2011; Vincenz-Donnelly and Hipp, 2017). BiP belongs to the highly conserved Hsp chaperone family and shares about 60% of its sequence identity with E. coli Hsp (DnaK) and human cytosolic Hsp70s (Wang et al, 2017). BiP and other Hsp70s have identical domain organization (Behnke et al, 2015) (Figure 1): They consist of a C-terminal substrate-binding domain (SBD) and an N-terminal nucleotide-binding domain (NDB), which are attached by a highly conserved hydrophobic linker. The SBD, which in turn, is made of a b-sandwich domain (bSBD) and a a-helical lid (aLid), binds to unfolded protein substrates, while the NBD is an ATPase that regulates the affinity of substrate binding to the SBD by using energy from ATP hydrolysis (Mayer et al, 2001; Mayer, 2013)

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Results

Conclusion