Showing papers by "Tania A. Baker published in 2022"
TL;DR: In this article , high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes are presented, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered.
Abstract: Abstract ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here we present high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze the protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated, tucked away from the channel and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies show additional structures and functions for key AAA+ elements. Pore-loop tucking may be used broadly by AAA+ unfoldases, for example, during enzyme pausing/unloading.
3 citations
TL;DR: In this paper , the authors demonstrate that FtsH degrades cyclopropane fatty acid (CFA) synthase, whose synthesis is induced upon nutrient deprivation and entry into stationary phase.
Abstract: Targeted protein degradation plays important roles in stress responses in all cells. In E. coli, the membrane‐bound AAA+ FtsH protease degrades cytoplasmic and membrane proteins. Here, we demonstrate that FtsH degrades cyclopropane fatty acid (CFA) synthase, whose synthesis is induced upon nutrient deprivation and entry into stationary phase. We find that neither the disordered N‐terminal residues nor the structured C‐terminal residues of the kinetically stable CFA‐synthase dimer are required for FtsH recognition and degradation. Experiments with fusion proteins support a model in which an internal degron mediates FtsH recognition as a prelude to unfolding and proteolysis. These findings elucidate the terminal step in the life cycle of CFA synthase and provide new insight into FtsH function.
2 citations
TL;DR: Cryo-EM structures of E. coli ClpXP, a AAA+ protease, are presented, which reveal that the axial channel of ClpX is closed prior to the binding and subsequent translocation of a protein substrate, indicating that channel closure contributes to increased degradation specificity.
Abstract: Intracellular proteases must be specific to avoid degrading the wrong proteins. Here, we present cryo-EM structures of E. coli ClpXP, a AAA+ protease, which reveal that the axial channel of ClpX is closed prior to the binding and subsequent translocation of a protein substrate. An open-channel ClpX mutation stimulates degradation of casein, a non-specific substrate, indicating that channel closure contributes to increased degradation specificity. We demonstrate that ClpX activates ClpP cleavage of a degron-free decapeptide by a channel-independent mechanism, in which the peptide substrate appears to pass through a symmetry mismatched gap in the interface between ClpX and ClpP before entering the degradation chamber via the axial portal of ClpP. The peptide products of ClpXP protein degradation are likely to exit the chamber by the reverse route.
1 citations
TL;DR: In this article , the SspB adaptor is shown to interact with the degradation tag of a GFP-ssrA substrate and with ClpXP in E. coli.
Abstract: 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. SIGNIFICANCE Intercellular 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.
TL;DR: Results using peptide‐array experiments, mutant DHFR proteins, and fusion proteins suggest that FtsH recognizes an internal sequence in a species of DHFR that is partially unfolded, indicating that its degradation capacity is broader than previously reported.
Abstract: AAA+ proteolytic machines play essential roles in maintaining and rebalancing the cellular proteome in response to stress, developmental cues, and environmental changes. Of the five AAA+ proteases in Escherichia coli, FtsH is unique in its attachment to the inner membrane and its function in degrading both membrane and cytosolic proteins. E. coli dihydrofolate reductase (DHFR) is a stable and biophysically well‐characterized protein, which a previous study found resisted FtsH degradation despite the presence of an ssrA degron. By contrast, we find that FtsH degrades DHFR fused to a long peptide linker and ssrA tag. Surprisingly, we also find that FtsH degrades DHFR with shorter linkers and ssrA tag, and without any linker or tag. Thus, FtsH must be able to recognize a sequence element or elements within DHFR. We find that FtsH degradation of DHFR is noncanonical in the sense that it does not rely upon recognition of an unstructured polypeptide at or near the N‐terminus or C‐terminus of the substrate. Results using peptide‐array experiments, mutant DHFR proteins, and fusion proteins suggest that FtsH recognizes an internal sequence in a species of DHFR that is partially unfolded. Overall, our findings provide insight into substrate recognition by FtsH and indicate that its degradation capacity is broader than previously reported.