Detection of dynamic protein-protein interactions within complexes and networks remains a challenging task. Here, we show on 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 to co-immunoprecipitation without cross-linker revealing unprecedented insight into intracellular ClpXP networks.
Fux, A., Korotkov, V.S., Schneider, M., Antes, I., Sieber, S.A., "Chemical cross-linking enables drafting ClpXP proximity maps and taking snapshots of in situ interaction networks ", Cell Chem. Biol. 2018, accepted
Reproduced with permission.