Dataset Information

Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (?(S)) by ClpXP.

ABSTRACT: In Escherichia coli, ?(S) is the master regulator of the general stress response. The level of ?(S) changes in response to multiple stress conditions and it is regulated at many levels including protein turnover. In the absence of stress, ?(S) is rapidly degraded by the AAA+ protease, ClpXP in a regulated manner that depends on the adaptor protein RssB. This two-component response regulator mediates the recognition of ?(S) and its delivery to ClpXP. The turnover of ?(S) however, can be inhibited in a stress specific manner, by one of three anti-adaptor proteins. Each anti-adaptor binds to RssB and inhibits its activity, but how this is achieved is not fully understood at a molecular level. Here, we describe details of the interaction between each anti-adaptor and RssB that leads to the stabilization of ?(S). By defining the domains of RssB using partial proteolysis we demonstrate that each anti-adaptor uses a distinct mode of binding to inhibit RssB activity. IraD docks specifically to the N-terminal domain of RssB, IraP interacts primarily with the C-terminal domain, while IraM interacts with both domains. Despite these differences in binding, we propose that docking of each anti-adaptor induces a conformational change in RssB, which resembles the inactive dimer of RssB. This dimer-like state of RssB not only prevents substrate binding but also triggers substrate release from a pre-bound complex.

SUBMITTER: Micevski D

PROVIDER: S-EPMC4428439 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP.

Micevski Dimce D Zammit Jessica E JE Truscott Kaye N KN Dougan David A DA

Frontiers in molecular biosciences 20150423

In Escherichia coli, σ(S) is the master regulator of the general stress response. The level of σ(S) changes in response to multiple stress conditions and it is regulated at many levels including protein turnover. In the absence of stress, σ(S) is rapidly degraded by the AAA+ protease, ClpXP in a regulated manner that depends on the adaptor protein RssB. This two-component response regulator mediates the recognition of σ(S) and its delivery to ClpXP. The turnover of σ(S) however, can be inhibited ...[more]

PMID: 25988182

Similar Datasets

Project description:UnlabelledRpoS (σ(S)), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular σ(S) concentration is critical for appropriately scaled σ(S)-dependent gene expression. One way to maintain appropriate levels of σ(S) is to regulate its stability. Indeed, σ(S) degradation is catalyzed by the ClpXP protease and the recognition of σ(S) by ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist in Escherichia coli K-12; each interacts with RssB and inhibits RssB activity under different stress conditions, thereby stabilizing σ(S). Unlike K-12, some E. coli isolates, including uropathogenic E. coli strain CFT073, show comparable cellular levels of σ(S) during the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of σ(S) levels among E. coli strains. Here, we describe IraL, an RssB anti-adaptor that stabilizes σ(S) during logarithmic phase growth in CFT073 and other E. coli and Shigella strains. By immunoblot analyses, we show that IraL affects the levels and stability of σ(S) during logarithmic phase growth. By computational and PCR-based analyses, we reveal that iraL is found in many E. coli pathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found in E. coli species and suggest that differences in the regulation of σ(S) levels may contribute to host and niche specificity in pathogenic and nonpathogenic E. coli strains.ImportanceBacteria must cope with a variety of environmental conditions in order to survive. RpoS (σ(S)), the general stress response sigma factor, directs the expression of many genes under stressful conditions in both pathogenic and nonpathogenic Escherichia coli strains. The regulation of σ(S) levels and activity allows appropriately scaled σ(S)-dependent gene expression. Here, we describe IraL, an RssB anti-adaptor that, unlike previously described anti-adaptors, stabilizes σ(S) during the logarithmic growth phase in the absence of additional stress. We also demonstrate that iraL is found in a large number of E. coli and Shigella isolates. These data suggest that strains containing iraL are able to initiate σ(S)-dependent gene expression under conditions under which strains without iraL cannot. Therefore, IraL-mediated σ(S) stabilization may contribute to host and niche specificity in E. coli.

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

Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (?(S)) by ClpXP.

Publications

Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σ(S)) by ClpXP.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets