TY  - INPR
A1  - Kahraman, Kerem
A1  - Robson, Scott A.
A1  - Göcenler, Oktay
A1  - Yenici, Cansu M.
A1  - Tozkoparan, Cansu D.
A1  - Klein, Jennifer M.
A1  - Haas, Arthur L.
A1  - Ziarek, Joshua J.
A1  - Dağ, Çağdaş
T1  - Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: a combined SAXS and NMR study
T2  - bioRxiv
N2  - 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.
KW  - ISGylation
KW  - ubiquitination
KW  - E2 enzyme
KW  - TRACT
KW  - oligomerization
Y1  - 2023
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/83017
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-830172
UR  - https://www.biorxiv.org/content/10.1101/2023.04.13.536743v4
IS  - 2023.04.13.536743 Version 4
PB  - bioRxiv
ER  -