The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery
Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in organisms ranging from simple bacteria to humans. In E. coli and many other proteobacteria, the SspB adaptor assists ClpXP in degrading ssrA-tagged polypeptides produced as a consequence of tmRNA-mediated ribosome rescue. By tethering these incomplete ssrA-tagged proteins to ClpXP, SspB facilitates their efficient degradation at low substrate concentrations. How this process occurs structurally is unknown. Here, we present a cryo-EM structure of the SspB adaptor bound to a GFP-ssrA substrate and to ClpXP. This structure provides evidence for simultaneous contacts of SspB and ClpX with the ssrA tag within the tethering complex, allowing direct substrate handoff concomitant with the initiation of substrate translocation. Furthermore, our structures reveal that binding of the substrate*adaptor complex induces unexpected conformational changes within the spiral structure of the AAA+ ClpX hexamer and its interaction with the ClpP tetradecamer. SIGNIFICANCEIntercellular proteases, including ClpXP, degrade damaged or unneeded proteins. Peptide tags allow specific protein substrates to be recognized by the ClpX unfoldase/translocase component of ClpXP and by an adaptor, SspB, which tethers itself to ClpX and enhances ClpXP degradation of the tagged protein. Our cryo-EM structure of ClpXP bound to SspB and a tagged substrate shows that SspB and ClpX simultaneously contact the degradation tag and reveal changes in the structure of ClpX and its interaction with ClpP. These structural changes appear to be a prelude to an initial ClpX translocation step that pulls the substrate away from SspB and initiates degradation by allowing substrate unfolding and further translocation of the unfolded substrate into the proteolytic chamber of ClpP.