Communication & coordination between components of the ClpAP degradation machine
AAA+ (ATPases associated with diverse cellular activities) proteases are present in all kingdoms of life. These molecular machines perform energy-dependent regulated proteolysis. The bacterial enzyme ClpA is a double-ring hexameric AAA+ unfoldase/translocase that functions with the tetradecameric Cl...
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| Format: | Thesis |
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Massachusetts Institute of Technology
2023
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| Online Access: | https://hdl.handle.net/1721.1/147731 https://orcid.org/0000-0003-0960-5009 |
| _version_ | 1826209837203062784 |
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| author | Zuromski, Kristin L. |
| author2 | Baker, Tania A. |
| author_facet | Baker, Tania A. Zuromski, Kristin L. |
| author_sort | Zuromski, Kristin L. |
| collection | MIT |
| description | AAA+ (ATPases associated with diverse cellular activities) proteases are present in all kingdoms of life. These molecular machines perform energy-dependent regulated proteolysis. The bacterial enzyme ClpA is a double-ring hexameric AAA+ unfoldase/translocase that functions with the tetradecameric ClpP peptidase to degrade proteins that are damaged, unneeded, or require degradation for regulation. ClpA has two distinct, stacked rings, termed D1 and D2, constructed from hexamerization of subunits each containing two AAA+ modules. ClpA’s twelve AAA+ modules hydrolyze ATP and participate in the overall degradation process, but how the modules in D1 and D2 work together to power ATP-dependent degradation is not well-understood. Further, the mechanisms governing ClpA’s dynamic interactions with its partner peptidase, ClpP and with its adaptor protein, ClpS, remain unclear. Here, I present experiments that interrogate the coordination between components of ClpAP(S) to elucidate how these multiple proteins work together to form an efficient, regulated protease.
In Chapter I, I provide an overview of AAA+ protein mechanism, with an emphasis on specific features of ClpA(PS) to lay a foundation for the following chapters. I introduce a ClpA subunit crosslinking strategy in Chapter II and use this method to examine how ATP hydrolysis is coordinated between (i) modules in each of the D1 and D2 rings, and (ii) between the two rings. In Chapter III, I probe the contributions of the conserved structural loops in the D1 and D2 rings that line ClpA’s central channel during ClpAP degradation. I also interrogate the substrate delivery mechanism by the ClpS adaptor in this chapter, revealing distinct roles for pore loops in D1 and D2 during this handoff. I describe a ClpA-ClpP crosslinking experiment in Chapter IV to test a structural hypothesis that ClpA must rotate on ClpP during substrate translocation. Finally, in Chapter V, I provide a broader context for how the results described in Chapters II, III, and IV improve the field’s understanding of the division of labor and coordination of mechanical work in the ClpAPS degradation machine and suggest future areas of study to further elucidate mechanistic aspects of ClpA and other AAA+ proteins. |
| first_indexed | 2024-09-23T14:32:10Z |
| format | Thesis |
| id | mit-1721.1/147731 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T14:32:10Z |
| publishDate | 2023 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1477312023-01-27T03:13:59Z Communication & coordination between components of the ClpAP degradation machine Zuromski, Kristin L. Baker, Tania A. Sauer, Robert T. Massachusetts Institute of Technology. Department of Chemistry AAA+ (ATPases associated with diverse cellular activities) proteases are present in all kingdoms of life. These molecular machines perform energy-dependent regulated proteolysis. The bacterial enzyme ClpA is a double-ring hexameric AAA+ unfoldase/translocase that functions with the tetradecameric ClpP peptidase to degrade proteins that are damaged, unneeded, or require degradation for regulation. ClpA has two distinct, stacked rings, termed D1 and D2, constructed from hexamerization of subunits each containing two AAA+ modules. ClpA’s twelve AAA+ modules hydrolyze ATP and participate in the overall degradation process, but how the modules in D1 and D2 work together to power ATP-dependent degradation is not well-understood. Further, the mechanisms governing ClpA’s dynamic interactions with its partner peptidase, ClpP and with its adaptor protein, ClpS, remain unclear. Here, I present experiments that interrogate the coordination between components of ClpAP(S) to elucidate how these multiple proteins work together to form an efficient, regulated protease. In Chapter I, I provide an overview of AAA+ protein mechanism, with an emphasis on specific features of ClpA(PS) to lay a foundation for the following chapters. I introduce a ClpA subunit crosslinking strategy in Chapter II and use this method to examine how ATP hydrolysis is coordinated between (i) modules in each of the D1 and D2 rings, and (ii) between the two rings. In Chapter III, I probe the contributions of the conserved structural loops in the D1 and D2 rings that line ClpA’s central channel during ClpAP degradation. I also interrogate the substrate delivery mechanism by the ClpS adaptor in this chapter, revealing distinct roles for pore loops in D1 and D2 during this handoff. I describe a ClpA-ClpP crosslinking experiment in Chapter IV to test a structural hypothesis that ClpA must rotate on ClpP during substrate translocation. Finally, in Chapter V, I provide a broader context for how the results described in Chapters II, III, and IV improve the field’s understanding of the division of labor and coordination of mechanical work in the ClpAPS degradation machine and suggest future areas of study to further elucidate mechanistic aspects of ClpA and other AAA+ proteins. Ph.D. 2023-01-26T14:46:30Z 2023-01-26T14:46:30Z 2021-06 2021-06-16T17:40:41.051Z Thesis https://hdl.handle.net/1721.1/147731 https://orcid.org/0000-0003-0960-5009 In Copyright - Educational Use Permitted Copyright MIT http://rightsstatements.org/page/InC-EDU/1.0/ application/pdf Massachusetts Institute of Technology |
| spellingShingle | Zuromski, Kristin L. Communication & coordination between components of the ClpAP degradation machine |
| title | Communication & coordination between components of the ClpAP degradation machine |
| title_full | Communication & coordination between components of the ClpAP degradation machine |
| title_fullStr | Communication & coordination between components of the ClpAP degradation machine |
| title_full_unstemmed | Communication & coordination between components of the ClpAP degradation machine |
| title_short | Communication & coordination between components of the ClpAP degradation machine |
| title_sort | communication coordination between components of the clpap degradation machine |
| url | https://hdl.handle.net/1721.1/147731 https://orcid.org/0000-0003-0960-5009 |
| work_keys_str_mv | AT zuromskikristinl communicationcoordinationbetweencomponentsoftheclpapdegradationmachine |