Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners
Abstract
:1. Introduction
2. Results
2.1. Characterization of SPOP MATH Binding Mechanism
2.2. Increasing Ionic Strength Weakens the Affinity of SPOP MATH to Its Substrates
2.3. Analyzing the Binding of SPOP MATH by Site-Directed Mutagenesis
3. Discussion
4. Materials and Methods
4.1. Protein Expression and Purification
4.2. Equilibrium Binding Experiments
4.3. Stopped-Flow Binding Experiments
4.4. Stopped-Flow Displacement Experiments
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Kohler, V.; Andréasson, C. Reversible Protein Assemblies in the Proteostasis Network in Health and Disease. Front. Mol. Biosci. 2023, 10, 1155521. [Google Scholar] [CrossRef] [PubMed]
- Wilson, M.R.; Satapathy, S.; Vendruscolo, M. Extracellular Protein Homeostasis in Neurodegenerative Diseases. Nat. Rev. Neurol. 2023, 19, 235–245. [Google Scholar] [CrossRef] [PubMed]
- Barbour, H.; Nkwe, N.S.; Estavoyer, B.; Messmer, C.; Gushul-Leclaire, M.; Villot, R.; Uriarte, M.; Boulay, K.; Hlayhel, S.; Farhat, B.; et al. An Inventory of Crosstalk between Ubiquitination and Other Post-Translational Modifications in Orchestrating Cellular Processes. iScience 2023, 26, 106276. [Google Scholar] [CrossRef] [PubMed]
- Chen, H.-Y.; Chen, R.-H. Cullin 3 Ubiquitin Ligases in Cancer Biology: Functions and Therapeutic Implications. Front. Oncol. 2016, 6, 113. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Skaar, J.R.; Pagan, J.K.; Pagano, M. SCF Ubiquitin Ligase-Targeted Therapies. Nat. Rev. Drug Discov. 2014, 13, 889–903. [Google Scholar] [CrossRef] [Green Version]
- Akhouri, V.; Majumder, S.; Gaikwad, A.B. The Emerging Insight into E3 Ligases as the Potential Therapeutic Target for Diabetic Kidney Disease. Life Sci. 2023, 321, 121643. [Google Scholar] [CrossRef]
- Sampson, C.; Wang, Q.; Otkur, W.; Zhao, H.; Lu, Y.; Liu, X.; Piao, H.-L. The Roles of E3 Ubiquitin Ligases in Cancer Progression and Targeted Therapy. Clin. Transl. Med. 2023, 13, e1204. [Google Scholar] [CrossRef]
- Suresh, K.; Mattern, M.; Goldberg, M.S.; Butt, T.R. The Ubiquitin Proteasome System as a Therapeutic Area in Parkinson’s Disease. NeuroMolecular Med. 2023. [Google Scholar] [CrossRef]
- Nagai, Y.; Kojima, T.; Muro, Y.; Hachiya, T.; Nishizawa, Y.; Wakabayashi, T.; Hagiwara, M. Identification of a Novel Nuclear Speckle-Type Protein, SPOP. FEBS Lett. 1997, 418, 23–26. [Google Scholar] [CrossRef] [Green Version]
- Takahashi, I.; Kameoka, Y.; Hashimoto, K. MacroH2A1.2 Binds the Nuclear Protein Spop. Biochim. Biophys. Acta 2002, 1591, 63–68. [Google Scholar] [CrossRef] [Green Version]
- Wei, X.; Fried, J.; Li, Y.; Hu, L.; Gao, M.; Zhang, S.; Xu, B. Functional Roles of Speckle-Type Poz (SPOP) Protein in Genomic Stability. J. Cancer 2018, 9, 3257–3262. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vivanco, I.; Sawyers, C.L. The Phosphatidylinositol 3-Kinase–AKT Pathway in Human Cancer. Nat. Rev. Cancer 2002, 2, 489–501. [Google Scholar] [CrossRef] [PubMed]
- Zhuang, M.; Calabrese, M.F.; Liu, J.; Waddell, M.B.; Nourse, A.; Hammel, M.; Miller, D.J.; Walden, H.; Duda, D.M.; Seyedin, S.N.; et al. Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases. Mol. Cell 2009, 36, 39–50. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Van Geersdaele, L.K.; Stead, M.A.; Harrison, C.M.; Carr, S.B.; Close, H.J.; Rosbrook, G.O.; Connell, S.D.; Wright, S.C. Structural Basis of High-Order Oligomerization of the Cullin-3 Adaptor SPOP. Acta Crystallogr. D Biol. Crystallogr. 2013, 69, 1677–1684. [Google Scholar] [CrossRef] [PubMed]
- Mani, R.-S. The Emerging Role of Speckle-Type POZ Protein (SPOP) in Cancer Development. Drug Discov. Today 2014, 19, 1498–1502. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yang, X.; Zhu, Q. SPOP in Cancer: Phenomena, Mechanisms and Its Role in Therapeutic Implications. Genes 2022, 13, 2051. [Google Scholar] [CrossRef] [PubMed]
- Malagrinò, F.; Troilo, F.; Bonetti, D.; Toto, A.; Gianni, S. Mapping the Allosteric Network within a SH3 Domain. Sci. Rep. 2019, 9, 8279. [Google Scholar] [CrossRef] [Green Version]
- Laursen, L.; Gianni, S.; Jemth, P. Dissecting Inter-Domain Cooperativity in the Folding of a Multi Domain Protein. J. Mol. Biol. 2021, 433, 167148. [Google Scholar] [CrossRef]
- Haq, S.R.; Chi, C.N.; Bach, A.; Dogan, J.; Engström, Å.; Hultqvist, G.; Karlsson, O.A.; Lundström, P.; Montemiglio, L.C.; Strømgaard, K.; et al. Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains. J. Am. Chem. Soc. 2012, 134, 599–605. [Google Scholar] [CrossRef] [Green Version]
- Fersht, A. Structure and Mechanism in Protein Science. In A Guide to Enzyme Catalysis and Protein Folding; W. H. Freeman: New York, NY, USA, 1999. [Google Scholar]
- Savino, C.; Montemiglio, L.C.; Sciara, G.; Miele, A.E.; Kendrew, S.G.; Jemth, P.; Gianni, S.; Vallone, B. Investigating the Structural Plasticity of a Cytochrome P450: Three-Dimensional Structures of P450 EryK and Binding to Its Physiological Substrate. J. Biol. Chem. 2009, 284, 29170–29179. [Google Scholar] [CrossRef] [Green Version]
- Pereira, C.S.; Teixeira, M.H.; Russell, D.A.; Hirst, J.; Arantes, G.M. Mechanism of Rotenone Binding to Respiratory Complex I Depends on Ligand Flexibility. Sci. Rep. 2023, 13, 6738. [Google Scholar] [CrossRef] [PubMed]
- Ahn, D.; Provasi, D.; Duc, N.M.; Xu, J.; Salas-Estrada, L.; Spasic, A.; Yun, M.W.; Kang, J.; Gim, D.; Lee, J.; et al. Gαs Slow Conformational Transition upon GTP Binding and a Novel Gαs Regulator. iScience 2023, 26, 106603. [Google Scholar] [CrossRef] [PubMed]
- Comez, D.; Glenn, J.; Anbuhl, S.M.; Heukers, R.; Smit, M.J.; Hill, S.J.; Kilpatrick, L.E. Fluorescently Tagged Nanobodies and NanoBRET to Study Ligand-Binding and Agonist-Induced Conformational Changes of Full-Length EGFR Expressed in Living Cells. Front. Immunol. 2022, 13, 1006718. [Google Scholar] [CrossRef] [PubMed]
- Chi, C.N.; Bach, A.; Engström, A.; Wang, H.; Strømgaard, K.; Gianni, S.; Jemth, P. A Sequential Binding Mechanism in a PDZ Domain. Biochemistry 2009, 48, 7089–7097. [Google Scholar] [CrossRef]
Equilibrium | Kinetics | |||
---|---|---|---|---|
KD (µM) | kon (µM−1 s−1) | koff (s−1) | KD (µM) | |
Puc | 1.4 ± 0.5 | 19 ± 1 | 26.1 ± 0.1 | 1.4 ± 0.4 |
MacroH2A | 1.6 ± 0.2 | 3 ± 2 | 21 ± 2 | 7.8 ± 0.4 |
PTEN | 4 ± 1 | 5.5 ± 0.5 | 113 ± 1 | 20.8 ± 0.4 |
MATH Variants | Puc | ||
---|---|---|---|
kon (µM−1 s−1) | koff (s−1) | KD (µM) | |
WT | 19 ± 1 | 26.1 ± 0.1 | 1.4 ± 0.4 |
R71A | 8.8 ± 0.5 | 34 ± 3 | 3.8 ± 0.1 |
Y88A | 14.4 ± 0.8 | 302 ± 5 | 21 ± 4 |
Y125A | 6.71 ± 0.02 | 78.0 ± 0.2 | 11.7 ± 0.2 |
K130A | 13 ± 2 | 123 ± 16 | 9.5 ± 0.1 |
D131A | 18.2 ± 0.1 | 241 ± 1 | 13.3 ± 0.2 |
W132A | N/A | N/A | N/A |
F134A | 9.4 ± 0.2 | 15 ± 1 | 1.7 ± 0.1 |
K135A | 6.3 ± 0.5 | 45.2 ± 0.4 | 7.2 ± 0.4 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Diop, A.; Pietrangeli, P.; Nardella, C.; Pennacchietti, V.; Pagano, L.; Toto, A.; Di Felice, M.; Di Matteo, S.; Marcocci, L.; Malagrinò, F.; et al. Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners. Int. J. Mol. Sci. 2023, 24, 10138. https://doi.org/10.3390/ijms241210138
Diop A, Pietrangeli P, Nardella C, Pennacchietti V, Pagano L, Toto A, Di Felice M, Di Matteo S, Marcocci L, Malagrinò F, et al. Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners. International Journal of Molecular Sciences. 2023; 24(12):10138. https://doi.org/10.3390/ijms241210138
Chicago/Turabian StyleDiop, Awa, Paola Pietrangeli, Caterina Nardella, Valeria Pennacchietti, Livia Pagano, Angelo Toto, Mariana Di Felice, Sara Di Matteo, Lucia Marcocci, Francesca Malagrinò, and et al. 2023. "Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners" International Journal of Molecular Sciences 24, no. 12: 10138. https://doi.org/10.3390/ijms241210138