Dataset Information

The AAA+ protease ClpXP can easily degrade a 31 and a 52-knotted protein.

ABSTRACT: Knots in proteins are hypothesized to make them resistant to enzymatic degradation by ATP-dependent proteases and recent studies have shown that whereas ClpXP can easily degrade a protein with a shallow 31 knot, it cannot degrade 52-knotted proteins if degradation is initiated at the C-terminus. Here, we present detailed studies of the degradation of both 31- and 52-knotted proteins by ClpXP using numerous constructs where proteins are tagged for degradation at both N- and C-termini. Our results confirm and extend earlier work and show that ClpXP can easily degrade a deeply 31-knotted protein. In contrast to recently published work on the degradation of 52-knotted proteins, our results show that the ClpXP machinery can also easily degrade these proteins. However, the degradation depends critically on the location of the degradation tag and the local stability near the tag. Our results are consistent with mechanisms in which either the knot simply slips along the polypeptide chain and falls off the free terminus, or one in which the tightened knot enters the translocation pore of ClpXP. Results of experiments on knotted protein fusions with a highly stable domain show partial degradation and the formation of degradation intermediates.

SUBMITTER: Sivertsson EM

PROVIDER: S-EPMC6382783 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Publications

The AAA+ protease ClpXP can easily degrade a 31 and a 52-knotted protein.

Sivertsson Elin M EM Jackson Sophie E SE Itzhaki Laura S LS

Scientific reports 20190220 1

Knots in proteins are hypothesized to make them resistant to enzymatic degradation by ATP-dependent proteases and recent studies have shown that whereas ClpXP can easily degrade a protein with a shallow 31 knot, it cannot degrade 52-knotted proteins if degradation is initiated at the C-terminus. Here, we present detailed studies of the degradation of both 31- and 52-knotted proteins by ClpXP using numerous constructs where proteins are tagged for degra ...[more]

PMID: 30787316

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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 AAA+ protease ClpXP can easily degrade a 31 and a 52-knotted protein.

Publications

The AAA+ protease ClpXP can easily degrade a 3<sub>1</sub> and a 5<sub>2</sub>-knotted protein.

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