Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease
ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates dire...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier BV
2022
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| Online Access: | https://hdl.handle.net/1721.1/146734 |
| _version_ | 1826203620249436160 |
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| author | Zuromski, Kristin L Kim, Sora Sauer, Robert T Baker, Tania A |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Zuromski, Kristin L Kim, Sora Sauer, Robert T Baker, Tania A |
| author_sort | Zuromski, Kristin L |
| collection | MIT |
| description | ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA's substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to "tug on" and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines. |
| first_indexed | 2024-09-23T12:40:10Z |
| format | Article |
| id | mit-1721.1/146734 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T12:40:10Z |
| publishDate | 2022 |
| publisher | Elsevier BV |
| record_format | dspace |
| spelling | mit-1721.1/1467342022-12-02T03:23:23Z Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease Zuromski, Kristin L Kim, Sora Sauer, Robert T Baker, Tania A Massachusetts Institute of Technology. Department of Biology ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA's substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to "tug on" and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines. 2022-12-01T18:49:48Z 2022-12-01T18:49:48Z 2021 2022-12-01T18:44:09Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/146734 Zuromski, Kristin L, Kim, Sora, Sauer, Robert T and Baker, Tania A. 2021. "Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease." Journal of Biological Chemistry, 297 (6). en 10.1016/J.JBC.2021.101407 Journal of Biological Chemistry Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier BV Elsevier |
| spellingShingle | Zuromski, Kristin L Kim, Sora Sauer, Robert T Baker, Tania A Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease |
| title | Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease |
| title_full | Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease |
| title_fullStr | Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease |
| title_full_unstemmed | Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease |
| title_short | Division of labor between the pore-1 loops of the D1 and D2 AAA+ rings coordinates substrate selectivity of the ClpAP protease |
| title_sort | division of labor between the pore 1 loops of the d1 and d2 aaa rings coordinates substrate selectivity of the clpap protease |
| url | https://hdl.handle.net/1721.1/146734 |
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