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E3 ubiquitin ligase RNF213 employs a non-canonical zinc finger active site and is allosterically regulated by ATP

View ORCID ProfileJuraj Ahel, View ORCID ProfileAdam Fletcher, View ORCID ProfileDaniel B. Grabarczyk, View ORCID ProfileElisabeth Roitinger, Luiza Deszcz, Anita Lehner, View ORCID ProfileSatpal Virdee, View ORCID ProfileTim Clausen
doi: https://doi.org/10.1101/2021.05.10.443411
Juraj Ahel
1Research Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria
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  • For correspondence: juraj.ahel@imp.ac.at adam.fletcher@glasgow.ac.uk s.s.virdee@dundee.ac.uk tim.clausen@imp.ac.at
Adam Fletcher
2MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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  • For correspondence: juraj.ahel@imp.ac.at adam.fletcher@glasgow.ac.uk s.s.virdee@dundee.ac.uk tim.clausen@imp.ac.at
Daniel B. Grabarczyk
1Research Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria
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Elisabeth Roitinger
3Institute of Molecular Biotechnology (IMBA), Vienna BioCenter, Vienna, Austria
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Luiza Deszcz
1Research Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria
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Anita Lehner
4Vienna BioCenter Core Facilities, Vienna BioCenter, Vienna, Austria.
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Satpal Virdee
2MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom
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  • For correspondence: juraj.ahel@imp.ac.at adam.fletcher@glasgow.ac.uk s.s.virdee@dundee.ac.uk tim.clausen@imp.ac.at
Tim Clausen
1Research Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria
5Medical University of Vienna, Vienna, Austria
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  • For correspondence: juraj.ahel@imp.ac.at adam.fletcher@glasgow.ac.uk s.s.virdee@dundee.ac.uk tim.clausen@imp.ac.at
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Abstract

RNF213 is a giant E3 ubiquitin ligase and a major susceptibility factor of Moyamoya disease, a cerebrovascular disorder that can result in stroke or death. In the cell, RNF213 is involved in lipid droplet formation, lipotoxicity, hypoxia, and NF-κB signaling, but its exact function in these processes is unclear. Structural characterization has revealed the presence of a dynein- like ATPase module and an unprecedented but poorly understood E3 module. Here, we demonstrate that RNF213 E3 activity is dependent on ATP binding, rather than ATP hydrolysis, and is particularly responsive to the ATP/ADP/AMP ratio. Biochemical and activity-based probe analyses identify a non-canonical zinc finger domain as the E3 active site, which utilizes the strictly conserved Cys4462, not involved in zinc coordination, as the reactive nucleophile. The cryo-EM structure of the trapped RNF213:E2∼Ub intermediate reveals RNF213 C-terminal domain as the E2 docking site, which positions the ubiquitin-loaded E2 proximal to the catalytic zinc finger, facilitating nucleophilic attack of Cys4462 on the E2∼Ub thioester. Our findings show that RNF213 represents an undescribed type of a transthiolation E3 enzyme and is regulated by adenine nucleotide concentration via its ATPase core, possibly allowing it to react to changing metabolic conditions in the cell.

Competing Interest Statement

S.V. is an author of a patent relating to the ABP technology and is also Founder and shareholder for of a biotech company focused on E3 ligases, which he receives consultancy revenue from.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 10, 2021.
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E3 ubiquitin ligase RNF213 employs a non-canonical zinc finger active site and is allosterically regulated by ATP
Juraj Ahel, Adam Fletcher, Daniel B. Grabarczyk, Elisabeth Roitinger, Luiza Deszcz, Anita Lehner, Satpal Virdee, Tim Clausen
bioRxiv 2021.05.10.443411; doi: https://doi.org/10.1101/2021.05.10.443411
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E3 ubiquitin ligase RNF213 employs a non-canonical zinc finger active site and is allosterically regulated by ATP
Juraj Ahel, Adam Fletcher, Daniel B. Grabarczyk, Elisabeth Roitinger, Luiza Deszcz, Anita Lehner, Satpal Virdee, Tim Clausen
bioRxiv 2021.05.10.443411; doi: https://doi.org/10.1101/2021.05.10.443411

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