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Cell Chem Biol. 2019 Jan 17;26(1):48-59.e7. doi: 10.1016/j.chembiol.2018.10.007. Epub 2018 Nov 8.

Chemical Cross-Linking Enables Drafting ClpXP Proximity Maps and Taking Snapshots of In Situ Interaction Networks.

Author information

1
Department of Chemistry, Chair of Organic Chemistry II, Center for Integrated Protein Science (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany.
2
Department of Biociences, Center for Integrated Protein Science (CIPSM), Technische Universität München, Emil-Erlenmeyer-Forum 8, 85354 Freising, Germany.
3
Department of Chemistry, Chair of Organic Chemistry II, Center for Integrated Protein Science (CIPSM), Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany. Electronic address: stephan.sieber@tum.de.

Abstract

Detection of dynamic protein-protein interactions within complexes and networks remains a challenging task. Here, we show by the example of the proteolytic ClpXP complex the utility of combined chemical cross-linking and mass spectrometry (XL-MS) to map interactions within ClpP and ClpX as well as across the enigmatic ClpX hexamer-ClpP heptamer interface. A few hot-spot lysines located in signature loops in ClpX were shown to be in proximity to several structural regions of ClpP providing an initial draft of the ClpX-ClpP interaction. Application of XL-MS further confirmed that Listeria monocytogenes ClpX interacts with the heterooligomeric ClpP1/2 complex solely via the ClpP2 apical site. Moreover, cellular interaction networks of human and bacterial proteases were elucidated via in situ chemical cross-linking followed by an antibody-based pull-down against ClpP. A subsequent mass spectrometric analysis demonstrated an up to 3-fold higher coverage compared with co-immunoprecipitation without cross-linker revealing unprecedented insight into intracellular ClpXP networks.

KEYWORDS:

ClpP; ClpX; co-immunoprecipitation; cross-linking; mass spectrometry; protease; proteomics

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