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Abstract The AAA protease FtsH associates with HflK/C subunits to form a megadalton-size complex that spans the inner membrane and extends into the periplasm ofE. coli. How this bacterial complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following the overproduction of protein components, recent cryo-EM structures showed symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit the degradation of membrane-embedded substrates. Here, we present structures of native protein complexes, in which HflK/C instead forms an asymmetric nautilus-shaped assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. Membrane curvature in our FtsH•HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid scramblase activity and possibly with FtsH’s function in the degradation of membrane-embedded proteins. 

Abstract AAA+ proteases degrade intracellular proteins in a highly specific manner.E. coliClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed inE. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity. 

Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in bacteria and eukaryotic organelles of bacterial origin. In  Escherichia 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 structure reveals 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.