Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: a combined SAXS and NMR study

  • Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.

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Metadaten
Author:Kerem Kahraman, Scott A. RobsonORCiD, Oktay GöcenlerORCiD, Cansu M. YeniciORCiD, Cansu D. TozkoparanORCiD, Jennifer M. Klein, Arthur L. Haas, Joshua J. ZiarekORCiD, Çağdaş DağORCiD
URN:urn:nbn:de:hebis:30:3-830172
URL:https://www.biorxiv.org/content/10.1101/2023.04.13.536743v4
DOI:https://doi.org/10.1101/2023.04.13.536743
Parent Title (English):bioRxiv
Publisher:bioRxiv
Document Type:Preprint
Language:English
Year of Completion:2023
Year of first Publication:2023
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2024/04/15
Tag:E2 enzyme; ISGylation; TRACT; oligomerization; ubiquitination
Issue:2023.04.13.536743 Version 4
Edition:Version 4
Page Number:27
HeBIS-PPN:517296853
Institutes:Biochemie, Chemie und Pharmazie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Licence (German):License LogoCreative Commons - CC BY-NC-ND - Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International