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

摘要

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 thatListeria monocytogenesClpX interacts with the heterooligomeric ClpP1/2 complex solely via the ClpP2 apical site. Moreover,?cellular interaction networks of human and bacterial proteases were elucidated viain situchemical 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.