Kinetics characterization of ASXL1/2-mediated allosteric regulation of BAP1 deubiquitinase
Abstract BAP1 is a ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like protein ASXL2. Cancer-related mutations/deletions of BAP1 lead to loss-of-function either by directly targeting the catalytic (UCH) or ULD domains of BAP1, the latter disrupts binding to ASXL2, an obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of the domains involved in forming the enzymatically active complex are unknown. Here we investigate the molecular dynamics, kinetics and stoichiometry of these interactions. We demonstrate that the BAP1 and ASXL2 domain/proteins or protein complexes produced in either bacteria or baculovirus are structurally and functionally active. The interaction between BAP1 and ASXL2 is direct, specific, and stable to in vitro biochemical and biophysical manipulations as detected by isothermal titration calorimetry, GST association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 deubiquitinase activity. A stable ternary complex can be formed comprised of the BAP1-UCH, BAP1-ULD, and ASXL2-AB domains. Binding of the BAP1-ULD domain to the ASXL2-AB box is rapid, with fast association and slow dissociation rates. Stoichiometric analysis revealed that one molecule of the ULD domain directly interacts with one molecule of the AB Box. Real-time kinetics analysis of ULD/AB protein complex to the UCH domain of BAP1, based on SPR, indicated that formation of the ULD/AB complex with the UCH domain is a single-step event with fast association and slow dissociation rates. These structural and dynamic parameters implicate the possibility for future small-molecule approaches to reactivate latent wild-type UCH activity in BAP-mutant malignancies..
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Preprint| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
bioRxiv.org - (2021) vom: 15. Dez. Zur Gesamtaufnahme - year:2021 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Peng, Hongzhuang [VerfasserIn] |
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| Links: |
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| doi: |
10.1101/2020.02.24.962464 |
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| PPN (Katalog-ID): |
XBI00074509X |
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| 520 | |a Abstract BAP1 is a ubiquitin hydrolase whose deubiquitinase activity is mediated by polycomb group-like protein ASXL2. Cancer-related mutations/deletions of BAP1 lead to loss-of-function either by directly targeting the catalytic (UCH) or ULD domains of BAP1, the latter disrupts binding to ASXL2, an obligate partner for BAP1 enzymatic activity. However, the biochemical and biophysical properties of the domains involved in forming the enzymatically active complex are unknown. Here we investigate the molecular dynamics, kinetics and stoichiometry of these interactions. We demonstrate that the BAP1 and ASXL2 domain/proteins or protein complexes produced in either bacteria or baculovirus are structurally and functionally active. The interaction between BAP1 and ASXL2 is direct, specific, and stable to in vitro biochemical and biophysical manipulations as detected by isothermal titration calorimetry, GST association, and optical biosensor assays. Association of the ASXL2-AB box greatly stimulates BAP1 deubiquitinase activity. A stable ternary complex can be formed comprised of the BAP1-UCH, BAP1-ULD, and ASXL2-AB domains. Binding of the BAP1-ULD domain to the ASXL2-AB box is rapid, with fast association and slow dissociation rates. Stoichiometric analysis revealed that one molecule of the ULD domain directly interacts with one molecule of the AB Box. Real-time kinetics analysis of ULD/AB protein complex to the UCH domain of BAP1, based on SPR, indicated that formation of the ULD/AB complex with the UCH domain is a single-step event with fast association and slow dissociation rates. These structural and dynamic parameters implicate the possibility for future small-molecule approaches to reactivate latent wild-type UCH activity in BAP-mutant malignancies. | ||
| 700 | 1 | |a Cassel, Joel |e verfasserin |4 aut | |
| 700 | 1 | |a McCracken, Daniel S. |e verfasserin |4 aut | |
| 700 | 1 | |a Prokop, Jeremy W. |e verfasserin |4 aut | |
| 700 | 1 | |a Collop, Paul R. |e verfasserin |4 aut | |
| 700 | 1 | |a Polo, Alexander |e verfasserin |4 aut | |
| 700 | 1 | |a Joshi, Surbhi |e verfasserin |4 aut | |
| 700 | 1 | |a Mandell, Jacob P. |e verfasserin |4 aut | |
| 700 | 1 | |a Ayyanathan, Kasirajan |e verfasserin |4 aut | |
| 700 | 1 | |a Hinds, David |e verfasserin |4 aut | |
| 700 | 1 | |a Malkowicz, S. Bruce |e verfasserin |4 aut | |
| 700 | 1 | |a Harbour, J. William |e verfasserin |4 aut | |
| 700 | 1 | |a Bowcock, Anne M. |e verfasserin |4 aut | |
| 700 | 1 | |a Salvino, Joseph |e verfasserin |4 aut | |
| 700 | 1 | |a Kennedy, Eileen J. |e verfasserin |4 aut | |
| 700 | 1 | |a Testa, Joseph R. |e verfasserin |4 aut | |
| 700 | 1 | |a Rauscher, Frank J. |e verfasserin |4 aut | |
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