An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin
Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-pept...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Journal Article |
| Language: | English |
| Published: |
2020
|
| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/144645 |
| _version_ | 1826120987269136384 |
|---|---|
| author | Coufalova, Dominika Remnant, Lucy Hernychova, Lenka Muller, Petr Healy, Alan Kannan, Srinivasaraghavan Westwood, Nicholas Verma, Chandra Shekhar Vojtesek, Borek Hupp, Ted R. Houston, Douglas R. |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Coufalova, Dominika Remnant, Lucy Hernychova, Lenka Muller, Petr Healy, Alan Kannan, Srinivasaraghavan Westwood, Nicholas Verma, Chandra Shekhar Vojtesek, Borek Hupp, Ted R. Houston, Douglas R. |
| author_sort | Coufalova, Dominika |
| collection | NTU |
| description | Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit “rim” of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 forms a dominant oligomer stabilizing side chain. The monomeric ReptinY340A mutant protein exhibited increased activity to its partner protein AGR2 in an ELISA assay, further suggesting that hexamer formation can preclude certain protein interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the Y340A mutation attenuated deuterium suppression of Reptin in this motif in the presence of ligand. By contrast, the tyrosine motif of Reptin interacts with a shallower pocket in the hetero-oligomeric structure containing Pontin and HDX-MS revealed no obvious role of the Y340 side chain in stabilizing the Reptin-Pontin oligomer. Molecular dynamic simulations (MDS) rationalized how the Y340A mutation impacts upon a normally stabilizing inter-subunit amino acid contact. MDS also revealed how the D299N mutation can, by contrast, remove oligomer de-stabilizing contacts. These data suggest that the Reptin interactome can be regulated by a ligand dependent equilibrium between monomeric and hexameric forms through a hydrophobic inter-subunit protein-protein interaction motif bridged by Tyrosine-340. Significance: Discovering dynamic protein-protein interactions is a fundamental aim of research in the life sciences. An emerging view of protein-protein interactions in higher eukaryotes is that they are driven by small linear polypeptide sequences; the linear motif. We report on the use of linear-peptide motif screens to discover a relatively high affinity peptide-protein interaction for the AAA+ and pro-oncogenic protein Reptin. This peptide interaction site was shown to form a ‘hot-spot’ protein-protein interaction site, and validated to be important for ligand-induced oligomerization of the Reptin protein. These biochemical data provide a foundation to understand how single point mutations in Reptin can impact on its oligomerization and protein-protein interaction landscape. |
| first_indexed | 2024-10-01T05:25:24Z |
| format | Journal Article |
| id | ntu-10356/144645 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:25:24Z |
| publishDate | 2020 |
| record_format | dspace |
| spelling | ntu-10356/1446452023-02-28T17:01:37Z An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin Coufalova, Dominika Remnant, Lucy Hernychova, Lenka Muller, Petr Healy, Alan Kannan, Srinivasaraghavan Westwood, Nicholas Verma, Chandra Shekhar Vojtesek, Borek Hupp, Ted R. Houston, Douglas R. School of Biological Sciences Engineering::Bioengineering Reptin Protein Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit “rim” of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 forms a dominant oligomer stabilizing side chain. The monomeric ReptinY340A mutant protein exhibited increased activity to its partner protein AGR2 in an ELISA assay, further suggesting that hexamer formation can preclude certain protein interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the Y340A mutation attenuated deuterium suppression of Reptin in this motif in the presence of ligand. By contrast, the tyrosine motif of Reptin interacts with a shallower pocket in the hetero-oligomeric structure containing Pontin and HDX-MS revealed no obvious role of the Y340 side chain in stabilizing the Reptin-Pontin oligomer. Molecular dynamic simulations (MDS) rationalized how the Y340A mutation impacts upon a normally stabilizing inter-subunit amino acid contact. MDS also revealed how the D299N mutation can, by contrast, remove oligomer de-stabilizing contacts. These data suggest that the Reptin interactome can be regulated by a ligand dependent equilibrium between monomeric and hexameric forms through a hydrophobic inter-subunit protein-protein interaction motif bridged by Tyrosine-340. Significance: Discovering dynamic protein-protein interactions is a fundamental aim of research in the life sciences. An emerging view of protein-protein interactions in higher eukaryotes is that they are driven by small linear polypeptide sequences; the linear motif. We report on the use of linear-peptide motif screens to discover a relatively high affinity peptide-protein interaction for the AAA+ and pro-oncogenic protein Reptin. This peptide interaction site was shown to form a ‘hot-spot’ protein-protein interaction site, and validated to be important for ligand-induced oligomerization of the Reptin protein. These biochemical data provide a foundation to understand how single point mutations in Reptin can impact on its oligomerization and protein-protein interaction landscape. Agency for Science, Technology and Research (A*STAR) National Supercomputing Centre (NSCC) Singapore Accepted version The work was supported by: the Czech Science Foundation 16-20860S (PM, LH) and 16-07321S (BV, TH), the project MEYS–NPS I–LO1413, and MH CZ - DRO (MMCI, 00209805); the BBSRC RASORconsortium (BB/C511599/1; United Kingdom); Cancer Research UK(C21383/A6950); The International Centre for Cancer Vaccine Scienceproject carried out within the International Research Agendas pro-gramme of the Foundation for Polish Science co-financed by theEuropean Union under the European Regional Development Fund;A*STAR, Singapore and NSCC, Singapore. 2020-11-17T01:53:51Z 2020-11-17T01:53:51Z 2019 Journal Article Coufalova, D., Remnant, L., Hernychova, L., Muller, P., Healy, A., Kannan, S., . . . Houston, D. R. (2019). An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin. Journal of Proteomics, 199, 89-101. doi:10.1016/j.jprot.2019.02.012 1874-3919 https://hdl.handle.net/10356/144645 10.1016/j.jprot.2019.02.012 30862565 199 89 101 en Journal of Proteomics © 2019 Elsevier B.V. All rights reserved. This paper was published in Journal of Proteomics and is made available with permission of Elsevier B.V. application/pdf |
| spellingShingle | Engineering::Bioengineering Reptin Protein Coufalova, Dominika Remnant, Lucy Hernychova, Lenka Muller, Petr Healy, Alan Kannan, Srinivasaraghavan Westwood, Nicholas Verma, Chandra Shekhar Vojtesek, Borek Hupp, Ted R. Houston, Douglas R. An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin |
| title | An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin |
| title_full | An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin |
| title_fullStr | An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin |
| title_full_unstemmed | An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin |
| title_short | An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin |
| title_sort | inter subunit protein peptide interface that stabilizes the specific activity and oligomerization of the aaa chaperone reptin |
| topic | Engineering::Bioengineering Reptin Protein |
| url | https://hdl.handle.net/10356/144645 |
| work_keys_str_mv | AT coufalovadominika anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT remnantlucy anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT hernychovalenka anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT mullerpetr anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT healyalan anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT kannansrinivasaraghavan anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT westwoodnicholas anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT vermachandrashekhar anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT vojtesekborek anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT hupptedr anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT houstondouglasr anintersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT coufalovadominika intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT remnantlucy intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT hernychovalenka intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT mullerpetr intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT healyalan intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT kannansrinivasaraghavan intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT westwoodnicholas intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT vermachandrashekhar intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT vojtesekborek intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT hupptedr intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin AT houstondouglasr intersubunitproteinpeptideinterfacethatstabilizesthespecificactivityandoligomerizationoftheaaachaperonereptin |