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Stepwise unfolding of a ? barrel protein by the AAA+ ClpXP protease.

ABSTRACT: In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded ? barrel of superfolder GFP-ssrA involves extraction of the C-terminal ? strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.

SUBMITTER: Nager AR

PROVIDER: S-EPMC3184388 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease.

Nager Andrew R AR Baker Tania A TA Sauer Robert T RT

Journal of molecular biology 20110729 1

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentratio ...[more]

PMID: 21821046

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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

Stepwise unfolding of a ? barrel protein by the AAA+ ClpXP protease.

Publications

Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease.

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