Research Project: Isg15 Ekleyen Heet Tipi E3 Enzimi Here5in 3 Boyutlu Kristal Yapısının Belirlenmeesi ve E2 Enzimi Ubch8 İle Bağlanma Bölgesinin NMR S pektroskopisi İle Tayini
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Contributors
Funders
ID
TB.00537
Authors
Dağ, Çağdaş
Faculty Member
Publications
Inconsistent protein stability despite Pre-HECT domain helix: Unveiling variability in HECT ligases
(Bentham Science, 2025) Dağ, Çağdaş; Ceylan, Cansu Deniz Tozkoparan; Cansız, Cemre Sare; n2STAR (Koç University Nanofabrication and Nanocharacterization Center for Scientifc and Technological Advanced Research); KUISCID (Koç University İşbank Center for Infectious Diseases); Yes; Ceylan, Cansu Deniz Tozkoparan; Research Center
Introduction Ubiquitin and ubiquitin-like systems play crucial roles across a wide range of organisms, from simple to complex. Among the three enzyme-mediated post-translational modification (PTM) steps, the ligation step is the most critical. HERC5, a member of the HECT ligase family, is one of the three enzymes involved in the ISGylation system. However, the precise start points and lengths of the HECT domains in HECT ligases are still under debate.Method Some studies suggest the inclusion of an additional N-terminal alpha helix region within the HECT domain. To investigate the structural biology of the HECT domain of HERC5, we produced and purified various lengths of the HERC5 HECT domain using different fusion proteins. This approach allowed us to explore the role of the N-terminal alpha helix in the stability of the HECT domain. Our experiments successfully produced and purified HERC5 HECT domains of different lengths with various fusion proteins.Result The findings demonstrated that the N-terminal alpha-helix does not enhance the stability of the HECT domain. These results challenge the notion that the N-terminal alpha-helix should be generally included in the HECT domain across all HECT ligases.Conclusion The inclusion of this region within the HECT domain may not be appropriate for generalization, as it does not contribute to stability, contrary to some previous suggestions.
Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography
(Cell Press, 2022) Ayan, Esra; Büyükdağ, Cengizhan; Çakılkaya, Barış; Dağ, Çağdaş; Demirci, Hasan; Ertem, Fatma Betül; Göcenler, Oktay; Gül, Mehmet; Güven, Ömür; Johnson, Jerome Austin; Karakadıoğlu, Gözde Usta; Yüksel, Büşra; Dao, E. Han; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Hayes, Brandon; Liang, Mengning; Hunter, Mark S.; Batyuk, Alexander; Sierra, Raymond G.; Ketawala, Gihan; Botha, Sabine; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; KUISCID (Koç University İşbank Center for Infectious Diseases); Yes; College of Sciences; GRADUATE SCHOOL OF SCIENCES AND ENGINEERING; Research Center
The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography.
Characterizing the Monomer-Dimer Equilibrium of UbcH8/Ube2L6: a combined SAXS and NMR study
(American Chemical Society, 2024) Ceylan, Cansu Deniz Tozkoparan; Dağ, Çağdaş; Göcenler, Oktay; Kahraman, Kerem; Yenici, Cansu Müşerref; Robson, Scott A.; Klein, Jennifer M.; Dotsch, Volker; Elgin, Emine Sonay; Haas, Arthur L.; Ziarek, Joshua J.; Department of Molecular Biology and Genetics; n2STAR (Koç University Nanofabrication and Nanocharacterization Center for Scientifc and Technological Advanced Research); KUISCID (Koç University İşbank Center for Infectious Diseases); Graduate School of Sciences and Engineering; Yes; College of Sciences; GRADUATE SCHOOL OF SCIENCES AND ENGINEERING; Research Center
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.
