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

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

doi:10.1038/s41467-021-22596-0

Cited by 19 publications

(40 citation statements)

References 77 publications

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“…Enzymes that mediate protein NMPylation frequently have low affinity for their targets, necessitating covalent linkage of enzyme:substrate complexes for structural analysis ( 41 ). The modified nsp9 appears to be released from SARS-CoV-2 RdRp ( Supplementary Figure S3A ) and multi-round NMPylation of HCoV-229E nsp9 has also been reported ( 20 ).…”

Section: Resultsmentioning

confidence: 99%

NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain

Wang

Svetlov²,

Artsimovitch

2021

Nucleic Acids Research

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The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.

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Section: Resultsmentioning

confidence: 99%

NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain

Wang

Svetlov²,

Artsimovitch

2021

Nucleic Acids Research

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“…Whilst this manuscript was in revision we became aware of a crystal structure of BiP covalently bound to FICD (PDB 6ZMD ) 34 , likely that of a transient intermediate post-AMPylation state. This covalent complex possess a near-identical FICD(TPR)–BiP interface to the deAMPylation complexes presented here, which accounts for the consistent outcome of the mutagenesis carried out in both studies.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…( 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.…”

Section: Figurementioning

confidence: 99%

“…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 .…”

Section: Introductionmentioning

confidence: 99%

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

Virolainen

Søltoft

Pedersen

et al. 2022

IJMS

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The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-luminal domain produced in Escherichia coli. Information about potentially important features, such as structural motifs, modulator binding sites or other regulatory elements, is therefore missing for the approximately 100 N-terminal residues including the transmembrane region of FICD. Expressing and purifying the required quantity and quality of membrane proteins is demanding because of the low yields and poor stability often observed. Here, we produce full-length FICD by combining a Saccharomyces cerevisiae-based platform with green fluorescent protein (GFP) tagging to optimise the conditions for expression, solubilisation and purification. We subsequently employ these conditions to purify milligram quantities of His-tagged FICD per litre of culture, and show that the purified, detergent-solubilised membrane protein is an active deAMPylating enzyme. Our work provides a straightforward methodology for producing not only full-length FICD, but also other membrane proteins in S. cerevisiae for structural and biochemical characterisation.

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

Cited by 19 publications

References 77 publications

NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain

NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain

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

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

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