Characterizing the Monomer-Dimer Equilibrium of UbcH8/Ube2L6: A Combined SAXS and NMR Study

Kerem Kahraman; Scott A. Robson; Oktay Göcenler; Cansu M. Yenici; Cansu D. Tozkoparan Ceylan; Jennifer M. Klein; Volker Dötsch; Emine Sonay Elgin; Arthur L. Haas; Joshua J. Ziarek; Çağdaş Dağ

doi:10.1021/acsomega.4c03610

Characterizing the Monomer-Dimer Equilibrium of UbcH8/Ube2L6: A Combined SAXS and NMR Study

Kerem Kahraman, Scott A. Robson, Oktay Göcenler, Cansu M. Yenici, Cansu D. Tozkoparan Ceylan, Jennifer M. Klein, Volker Dötsch, Emine Sonay Elgin, Arthur L. Haas, Joshua J. Ziarek^*, Çağdaş Dağ^*

^*Corresponding author for this work

Pharmacology

Research output: Contribution to journal › Article › peer-review

Abstract

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain and is a conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a 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 crystallized in both monomeric and dimeric forms, but its functional state remains 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 N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a back-side dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at the E1 and E3 interfaces, providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques to provide structural information about proteins in solution.

Original language	English (US)
Journal	ACS Omega
DOIs	https://doi.org/10.1021/acsomega.4c03610
State	Accepted/In press - 2024

Funding

C.D. acknowledges support from TU\u0308BI\u0307TAK (Project Nos. 120Z594 and 122Z747). J.J.Z. acknowledges support from National Institutes of Health (Grant No: R35GM143054). The authors acknowledge the use of the services and facilities of nSTAR-Koc\u0327 University Nanofabrication and Nanocharacterization Center for Scientific and Technological Advanced Research. We extend our sincere gratitude to Turkish Airlines for their generous support in providing transportation for the research team, which was crucial for the successful completion of this study. Their assistance greatly facilitated our work and data collection efforts.

ASJC Scopus subject areas

General Chemistry
General Chemical Engineering

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@article{66b00bc2801d418b9338cdb5240a954d,

title = "Characterizing the Monomer-Dimer Equilibrium of UbcH8/Ube2L6: A Combined SAXS and NMR Study",

abstract = "Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain and is a conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a 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 crystallized in both monomeric and dimeric forms, but its functional state remains unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8{\textquoteright}s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a back-side dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at the E1 and E3 interfaces, providing hypotheses for the protein{\textquoteright}s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques to provide structural information about proteins in solution.",

author = "Kerem Kahraman and Robson, {Scott A.} and Oktay G{\"o}cenler and Yenici, {Cansu M.} and {Tozkoparan Ceylan}, {Cansu D.} and Klein, {Jennifer M.} and Volker D{\"o}tsch and Elgin, {Emine Sonay} and Haas, {Arthur L.} and Ziarek, {Joshua J.} and {\c C}ağda{\c s} Dağ",

note = "Publisher Copyright: {\textcopyright} 2024 The Authors. Published by American Chemical Society.",

year = "2024",

doi = "10.1021/acsomega.4c03610",

language = "English (US)",

journal = "ACS Omega",

issn = "2470-1343",

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T1 - Characterizing the Monomer-Dimer Equilibrium of UbcH8/Ube2L6

T2 - A Combined SAXS and NMR Study

AU - Kahraman, Kerem

AU - Robson, Scott A.

AU - Göcenler, Oktay

AU - Yenici, Cansu M.

AU - Tozkoparan Ceylan, Cansu D.

AU - Klein, Jennifer M.

AU - Dötsch, Volker

AU - Elgin, Emine Sonay

AU - Haas, Arthur L.

AU - Ziarek, Joshua J.

AU - Dağ, Çağdaş

PY - 2024

Y1 - 2024

N2 - Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain and is a conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a 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 crystallized in both monomeric and dimeric forms, but its functional state remains 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 N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a back-side dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at the E1 and E3 interfaces, providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques to provide structural information about proteins in solution.

AB - Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain and is a conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a 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 crystallized in both monomeric and dimeric forms, but its functional state remains 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 N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a back-side dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at the E1 and E3 interfaces, providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques to provide structural information about proteins in solution.

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DO - 10.1021/acsomega.4c03610

M3 - Article

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SN - 2470-1343

JO - ACS Omega

JF - ACS Omega

ER -

Characterizing the Monomer-Dimer Equilibrium of UbcH8/Ube2L6: A Combined SAXS and NMR Study

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