Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain
PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2023-07-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/88058 |
| _version_ | 1797785272932368384 |
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| author | Noah J Harris Meredith L Jenkins Sung-Eun Nam Manoj K Rathinaswamy Matthew AH Parson Harish Ranga-Prasad Udit Dalwadi Brandon E Moeller Eleanor Sheeky Scott D Hansen Calvin K Yip John E Burke |
| author_facet | Noah J Harris Meredith L Jenkins Sung-Eun Nam Manoj K Rathinaswamy Matthew AH Parson Harish Ranga-Prasad Udit Dalwadi Brandon E Moeller Eleanor Sheeky Scott D Hansen Calvin K Yip John E Burke |
| author_sort | Noah J Harris |
| collection | DOAJ |
| description | PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention. |
| first_indexed | 2024-03-13T00:52:41Z |
| format | Article |
| id | doaj.art-e0ae711fee134081811275437049e492 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-13T00:52:41Z |
| publishDate | 2023-07-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-e0ae711fee134081811275437049e4922023-07-07T12:11:01ZengeLife Sciences Publications LtdeLife2050-084X2023-07-011210.7554/eLife.88058Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domainNoah J Harris0Meredith L Jenkins1https://orcid.org/0000-0002-0685-5048Sung-Eun Nam2Manoj K Rathinaswamy3Matthew AH Parson4https://orcid.org/0000-0001-6270-559XHarish Ranga-Prasad5Udit Dalwadi6Brandon E Moeller7Eleanor Sheeky8https://orcid.org/0000-0002-1501-550XScott D Hansen9https://orcid.org/0000-0001-7005-6200Calvin K Yip10https://orcid.org/0000-0003-1779-9501John E Burke11https://orcid.org/0000-0001-7904-9859Department of Biochemistry and Microbiology, University of Victoria, Victoria, CanadaDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, CanadaDepartment of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, CanadaDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, CanadaDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, CanadaDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, CanadaDepartment of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, CanadaDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, CanadaDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, CanadaDepartment of Chemistry and Biochemistry, Institute of Molecular Biology, University of Oregon, Eugene, United StatesDepartment of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, CanadaDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, Canada; Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, CanadaPI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.https://elifesciences.org/articles/88058PIK3CGp101PIK3R5PI3KPI3KγHDX-MS |
| spellingShingle | Noah J Harris Meredith L Jenkins Sung-Eun Nam Manoj K Rathinaswamy Matthew AH Parson Harish Ranga-Prasad Udit Dalwadi Brandon E Moeller Eleanor Sheeky Scott D Hansen Calvin K Yip John E Burke Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain eLife PIK3CG p101 PIK3R5 PI3K PI3Kγ HDX-MS |
| title | Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain |
| title_full | Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain |
| title_fullStr | Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain |
| title_full_unstemmed | Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain |
| title_short | Allosteric activation or inhibition of PI3Kγ mediated through conformational changes in the p110γ helical domain |
| title_sort | allosteric activation or inhibition of pi3kγ mediated through conformational changes in the p110γ helical domain |
| topic | PIK3CG p101 PIK3R5 PI3K PI3Kγ HDX-MS |
| url | https://elifesciences.org/articles/88058 |
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