Structural basis for inhibition of a response regulator of σ<sup>S</sup> stability by a ClpXP antiadaptor

Dorich, Victoria; Brugger, Christiane; Tripathi, Arti; Hoskins, Joel R.; Tong, Song; Suhanovsky, Margaret M.; Sastry, Amita; Wickner, Sue; Gottesman, Susan; Deaconescu, Alexandra M.

doi:10.1101/gad.320168.118

Cited by 23 publications

(57 citation statements)

References 62 publications

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“…The structure of an RssB homolog has been described (PDB code 3EQ2). This protein differs from RssB in having all the sites for an active phosphatase in the C-terminal domain (126) and a linker that appears to be significantly more extended than that found in E. coli RssB (95). Intriguingly, this protein, PA2798 in P. aeruginosa PA01, is found next to and likely translationally coupled with a protein annotated as an anti-anti-sigma, PA2797.…”

Section: General Stress Responses In Other Bacteria: Many Variationsmentioning

confidence: 92%

“…Recently, a structure of IraD in complex with RssB has been determined and shows IraD binding to and presumably stabilizing a "closed" collapsed form of RssB, with both domains of RssB bound by IraD (Fig. 4) (95). Further understanding of how other anti-adaptors inhibit RssB and how RssB delivers RpoS to the protease await future structures.…”

Section: Regulated Proteolysis Of Rposmentioning

confidence: 99%

“…4B, a modification of a figure used in Ref. 95, and I thank K. Ramamurthi and J. Chen for comments on the manuscript.…”

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confidence: 99%

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Trouble is coming: Signaling pathways that regulate general stress responses in bacteria

Gottesman

2019

Journal of Biological Chemistry

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Bacteria can rapidly and reversibly respond to changing environments via complex transcriptional and post-transcriptional regulatory mechanisms. Many of these adaptations are specific, with the regulatory output tailored to the inducing signal (for instance, repairing damage to cell components or improving acquisition and use of growth-limiting nutrients). However, the general stress response, activated in bacterial cells entering stationary phase or subjected to nutrient depletion or cellular damage, is unique in that its common, broad output is induced in response to many different signals. In many different bacteria, the key regulator for the general stress response is a specialized sigma factor, the promoter specificity subunit of RNA polymerase. The availability or activity of the sigma factor is regulated by complex regulatory circuits, the majority of which are post-transcriptional. In Escherichia coli, multiple small regulatory RNAs, each made in response to a different signal, positively regulate translation of the general stress response sigma factor RpoS. Stability of RpoS is regulated by multiple anti-adaptor proteins that are also synthesized in response to different signals. In this review, the modes of signaling to and levels of regulation of the E. coli general stress response are discussed. They are also used as a basis for comparison with the general stress response in other bacteria with the aim of extracting key principles that are common among different species and highlighting important unanswered questions.

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Section: General Stress Responses In Other Bacteria: Many Variationsmentioning

confidence: 92%

Section: Regulated Proteolysis Of Rposmentioning

confidence: 99%

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Trouble is coming: Signaling pathways that regulate general stress responses in bacteria

Gottesman

2019

Journal of Biological Chemistry

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183

133

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“…Each anti-adaptor is induced in response to a specific stress, and currently three anti-adaptor proteins have been identified. Although the precise mechanism of action of these anti-adaptors remains unclear, all are proposed to inhibit σ s turnover via direct interaction with RssB [22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Insight into the RssB-Mediated Recognition and Delivery of σs to the AAA+ Protease, ClpXP

et al. 2020

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In Escherichia coli, SigmaS (σS) is the master regulator of the general stress response. The cellular levels of σS are controlled by transcription, translation and protein stability. The turnover of σS, 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 σS 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 σS). Together with our biochemical analysis we propose a model for the recognition and delivery of σS by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (βα)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of σS, much of the direct binding to σS occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-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 α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σS interaction, as σS binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σS, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σS at a distal site.

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“…In the single available structure of a PP2C phosphatase bound to its substrate protein (Hab1 bound to SnRK2), SnRK2 makes direct contacts with the Hab1 switch and to a variable region (termed "the flap") that packs against the switch (Soon et al, 2012). Regulatory domains from diverse PP2C phosphatases similarly pack against the switch (Bradshaw et al, 2017;Dorich et al, 2019;Vassylyev and Symersky, 2007;Zhang et al, 2013). For different phosphatases, this coupling could be used to recruit substrate when the phosphatase is in its active conformation or to ensure that the phosphatase only adopts the active conformation when bound to the cognate substrate.…”

Section: Discussionmentioning

confidence: 99%

A conserved regulatory switch controls phosphatase activity and specificity

Ho¹,

Bradshaw²

2019

Preprint

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Protein phosphatases must be regulated and specific for their substrates; how this control is achieved is critical for signaling by reversible phosphorylation. We recently reported the discovery of a regulatory switch that controls the activity of the PP2C family serine/threonine phosphatase SpoIIE from the bacterium Bacillus subtilis. This regulatory switch activates SpoIIE during spore development by forming the catalytically essential metal-binding site that is conserved across the PP2C family. We hypothesized that this switch is a conserved platform for regulating other PP2C phosphatases. An orthologous phosphatase from B. subtilis, RsbU, is activated under stress conditions and responds to different signals and acts on a different phosphoprotein substrate than SpoIIE. Using a combination of biochemical and genetic approaches, we find that broad features of the regulatory mechanism are conserved between SpoIIE and RsbU but that each phosphatase has adapted its response to be most appropriate for the distinct biological outputs it controls. In both cases, the switch accomplishes this by integrating substrate binding and recognition with regulatory inputs to control metal cofactor binding and catalysis. Thus, the switch is a conserved and mechanistically flexible regulatory platform that controls phosphatase activity and substrate specificity.

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Structural basis for inhibition of a response regulator of σ^S stability by a ClpXP antiadaptor

Cited by 23 publications

References 62 publications

Trouble is coming: Signaling pathways that regulate general stress responses in bacteria

Trouble is coming: Signaling pathways that regulate general stress responses in bacteria

Insight into the RssB-Mediated Recognition and Delivery of σs to the AAA+ Protease, ClpXP

A conserved regulatory switch controls phosphatase activity and specificity

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Structural basis for inhibition of a response regulator of σS stability by a ClpXP antiadaptor

Cited by 23 publications

References 62 publications

Trouble is coming: Signaling pathways that regulate general stress responses in bacteria

Trouble is coming: Signaling pathways that regulate general stress responses in bacteria

Insight into the RssB-Mediated Recognition and Delivery of σs to the AAA+ Protease, ClpXP

A conserved regulatory switch controls phosphatase activity and specificity

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Structural basis for inhibition of a response regulator of σ^S stability by a ClpXP antiadaptor