Unveiling the Essential Role of Arkadia’s Non-RING Elements in the Ubiquitination Process
Abstract
:1. Introduction
2. Results and Discussion
2.1. The RING Domain of Arkadia Is Not Sufficient for Enzymatic Activity
2.2. Non-RING Elements Present in Ark 119 aa Polypeptide Enhance E2-E3 Interaction
2.3. Arkadia Isoform 1 Does Not Bind Ubiquitin
2.4. Screening Arkadia’s Non-RING Sequence That Is Crucial for E2 Interaction and E3 Ligation Activity
2.5. NRGA and TIER Segments Implication in E2 Interaction and Ubiquitination
3. Materials and Methods
3.1. Construct Design
3.2. Constructs, Protein Expression and Purification
3.3. Isothermal Titration Calorimetry (ITC)
3.4. Auto-Ubiquitination Assays
3.5. Synthesis of E2-Ub Conjugate
3.6. Oxyester Hydrolysis Assays
3.7. NMR Titration
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Protein | Kd (μΜ) | N | ΔH (Kcal/mol) | −TΔS (Kcal/mol) | ΔG (Kcal/mol) |
---|---|---|---|---|---|
Arkadia 68 aa | 32 | 1 | 3.1 | −9.3 | −6.14 |
Arkadia 119 aa | 2.7 | 0.6 * | −4.7 | −2.9 | −7.61 |
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Birkou, M.; Delegkou, G.N.; Marousis, K.D.; Fragkaki, N.; Toro, T.; Episkopou, V.; Spyroulias, G.A. Unveiling the Essential Role of Arkadia’s Non-RING Elements in the Ubiquitination Process. Int. J. Mol. Sci. 2022, 23, 10585. https://doi.org/10.3390/ijms231810585
Birkou M, Delegkou GN, Marousis KD, Fragkaki N, Toro T, Episkopou V, Spyroulias GA. Unveiling the Essential Role of Arkadia’s Non-RING Elements in the Ubiquitination Process. International Journal of Molecular Sciences. 2022; 23(18):10585. https://doi.org/10.3390/ijms231810585
Chicago/Turabian StyleBirkou, Maria, Georgia N. Delegkou, Konstantinos D. Marousis, Nefeli Fragkaki, Tamara Toro, Vasso Episkopou, and Georgios A. Spyroulias. 2022. "Unveiling the Essential Role of Arkadia’s Non-RING Elements in the Ubiquitination Process" International Journal of Molecular Sciences 23, no. 18: 10585. https://doi.org/10.3390/ijms231810585