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The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery.

ABSTRACT: 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.

SUBMITTER: Ghanbarpour A

PROVIDER: S-EPMC9963277 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery.

Ghanbarpour Alireza A Fei Xue X Baker Tania A TA Davis Joseph H JH Sauer Robert T RT

Proceedings of the National Academy of Sciences of the United States of America 20230202 6

Energy-dependent protein degradation by the AAA+ ClpXP protease helps maintain protein homeostasis in bacteria and eukaryotic organelles of bacterial origin. In <i>Escherichia coli</i> 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 proces ...[more]

PMID: 36730206

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Project description:In Escherichia coli, SigmaS (?S) is the master regulator of the general stress response. The cellular levels of ?S are controlled by transcription, translation and protein stability. The turnover of ?S, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)-which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of ?S recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssBN and RssBC) and the SAXS analysis of full-length RssB (both free and in complex with ?S). Together with our biochemical analysis we propose a model for the recognition and delivery of ?S by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (??)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of ?S, much of the direct binding to ?S occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a ?-sandwich fold composed of two sheets surrounded by ?-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a "closed" state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the ?5 helix of the N-terminal domain. Importantly, this residue is not directly involved in ?S interaction, as ?S binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of ?S, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the ?5 helix) plays a critical role in driving interaction with ?S at a distal site.

Dataset Information

The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery.

Publications

The SspB adaptor drives structural changes in the AAA+ ClpXP protease during ssrA-tagged substrate delivery.

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