Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD

Joel Fauser,Christoph Krisp,Martin Zacharias,Dorothea Höpfner,Aymelt Itzen,Christian Hedberg,Danial Pourjafar-Dehkordi,Gesa König,Christian Pett,Burak Gulen,Matthias J Feige,Vivian Pogenberg,Hartmut Schlüter

doi:10.1038/s41467-021-22596-0

Abstract

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.

Highlights

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the heat shock protein (Hsp)[70] chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ERresident filamentation induced by cyclic-AMP (Fic)-enzyme FICD/HYPE
We focused on charged aas that are located at the surface of the substrate-binding domain (SBD) and across the Fic domain, since these positions may interact with FICD in solution even though such contacts were not detected by crystallography
We validated the contribution of FICD’s TPR motifs to the recognition of BiP and provide an explanation for FICD preferentially targeting the domain-docked conformation of BiP19

Summary

Introduction

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp[70] chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ERresident Fic-enzyme FICD/HYPE. The nucleotide adenosine triphosphate (ATP) can bind to the NBD and is hydrolyzed to adenosine diphosphate (ADP) by the intrinsic ATPase activity with the concomitant release of phosphate During this ADP/ATP cycle, BiP adopts different conformations: in the ATP-bound state, BiP has low affinity to its substrates and its NBD and SBD are docked to each other with the conserved linker inserted into a specific pocket of the NBD5. In order to match ER folding capacities to short-term fluctuations of the unfolded protein load, two mechanisms directly regulate BiP: first, the oligomerization of BiP, and second, the covalent modification of BiP with an adenosine monophosphate (AMP) moiety[14,15,16] This process is a posttranslational modification, referred to as AMPylation, in which an AMP is transferred from ATP to the protein side chains[17,18]. It was demonstrated that AMPylation activity is conferred by monomeric FICD (induced by a single-point mutation L258D, disrupting dimerization) despite the integrity of the inhibitory motif, suggesting the monomeric species as AMPylator in vivo[31,32]

Methods

Results

Conclusion