Mapping Hsp104 interactions using cross‐linking mass spectrometry
Molecular machines from the AAA+ (ATPases Associated with diverse cellular Activity) superfamily of protein disaggregases play important roles in protein folding, disaggregation and DNA processing. Recent cryo‐EM structures of AAA+ molecular machines have uncovered nuanced changes in their conformat...
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| Format: | Article |
| Language: | English |
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Wiley
2025-06-01
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| Series: | FEBS Open Bio |
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| Online Access: | https://doi.org/10.1002/2211-5463.70007 |
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| author | Kinga Westphal Karolina Michalska Andrzej Joachimiak Lukasz A. Joachimiak |
| author_facet | Kinga Westphal Karolina Michalska Andrzej Joachimiak Lukasz A. Joachimiak |
| author_sort | Kinga Westphal |
| collection | DOAJ |
| description | Molecular machines from the AAA+ (ATPases Associated with diverse cellular Activity) superfamily of protein disaggregases play important roles in protein folding, disaggregation and DNA processing. Recent cryo‐EM structures of AAA+ molecular machines have uncovered nuanced changes in their conformation that underlie their specialized functions. Structural knowledge of these molecular machines in complex with substrates begins to explain their mechanism of activity. Here, we explore how cross‐linking mass spectrometry (XL‐MS) can be used to interpret changes in conformation induced by ATP in Hsp104 and how a substrate may interact with Hsp104. We applied a panel of cross‐linking reagents to produce cross‐linking maps of Hsp104 and interpret our data on previously determined X‐ray and cryo‐EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra‐subunit and inter‐subunit contacts within the hexameric homo‐oligomer. We identify cross‐links that break the asymmetry that is present in Hsp104 in an ATP‐hydrolysis competent conformation but is absent in an ATP‐hydrolysis‐defective mutant. Finally, we identify contacts between Hsp104 and a selected protein (proprotein convertase subtilisin/kexin type 9 PCSK9) to reveal contacts on the central channel of Hsp104 across the length of this protein indicating that we might have trapped interactions consistent with its translocation. Our simple and robust XL‐MS‐based experiments and methods help interpret how these molecular machines change conformation and bind to other proteins even in the context of homo‐oligomeric assemblies enabling coupling state‐of‐the‐art modeling approaches with XL‐MS. |
| format | Article |
| id | doaj-art-49ec219d81634ecb8c6a8ebd0f3a8142 |
| institution | Kabale University |
| issn | 2211-5463 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj-art-49ec219d81634ecb8c6a8ebd0f3a81422025-08-20T03:34:26ZengWileyFEBS Open Bio2211-54632025-06-0115692293910.1002/2211-5463.70007Mapping Hsp104 interactions using cross‐linking mass spectrometryKinga Westphal0Karolina Michalska1Andrzej Joachimiak2Lukasz A. Joachimiak3Center for Alzheimer's and Neurodegenerative Diseases Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center Dallas TX USACenter for Structural Genomics of Infectious Diseases, Consortium for Advanced Science and Engineering University of Chicago IL USACenter for Structural Genomics of Infectious Diseases, Consortium for Advanced Science and Engineering University of Chicago IL USACenter for Alzheimer's and Neurodegenerative Diseases Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center Dallas TX USAMolecular machines from the AAA+ (ATPases Associated with diverse cellular Activity) superfamily of protein disaggregases play important roles in protein folding, disaggregation and DNA processing. Recent cryo‐EM structures of AAA+ molecular machines have uncovered nuanced changes in their conformation that underlie their specialized functions. Structural knowledge of these molecular machines in complex with substrates begins to explain their mechanism of activity. Here, we explore how cross‐linking mass spectrometry (XL‐MS) can be used to interpret changes in conformation induced by ATP in Hsp104 and how a substrate may interact with Hsp104. We applied a panel of cross‐linking reagents to produce cross‐linking maps of Hsp104 and interpret our data on previously determined X‐ray and cryo‐EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra‐subunit and inter‐subunit contacts within the hexameric homo‐oligomer. We identify cross‐links that break the asymmetry that is present in Hsp104 in an ATP‐hydrolysis competent conformation but is absent in an ATP‐hydrolysis‐defective mutant. Finally, we identify contacts between Hsp104 and a selected protein (proprotein convertase subtilisin/kexin type 9 PCSK9) to reveal contacts on the central channel of Hsp104 across the length of this protein indicating that we might have trapped interactions consistent with its translocation. Our simple and robust XL‐MS‐based experiments and methods help interpret how these molecular machines change conformation and bind to other proteins even in the context of homo‐oligomeric assemblies enabling coupling state‐of‐the‐art modeling approaches with XL‐MS.https://doi.org/10.1002/2211-5463.70007Hsp104PCSK9protein–protein interactionsXL‐MS |
| spellingShingle | Kinga Westphal Karolina Michalska Andrzej Joachimiak Lukasz A. Joachimiak Mapping Hsp104 interactions using cross‐linking mass spectrometry FEBS Open Bio Hsp104 PCSK9 protein–protein interactions XL‐MS |
| title | Mapping Hsp104 interactions using cross‐linking mass spectrometry |
| title_full | Mapping Hsp104 interactions using cross‐linking mass spectrometry |
| title_fullStr | Mapping Hsp104 interactions using cross‐linking mass spectrometry |
| title_full_unstemmed | Mapping Hsp104 interactions using cross‐linking mass spectrometry |
| title_short | Mapping Hsp104 interactions using cross‐linking mass spectrometry |
| title_sort | mapping hsp104 interactions using cross linking mass spectrometry |
| topic | Hsp104 PCSK9 protein–protein interactions XL‐MS |
| url | https://doi.org/10.1002/2211-5463.70007 |
| work_keys_str_mv | AT kingawestphal mappinghsp104interactionsusingcrosslinkingmassspectrometry AT karolinamichalska mappinghsp104interactionsusingcrosslinkingmassspectrometry AT andrzejjoachimiak mappinghsp104interactionsusingcrosslinkingmassspectrometry AT lukaszajoachimiak mappinghsp104interactionsusingcrosslinkingmassspectrometry |