A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases
- Harvard University, United States
- University of York, United Kingdom
- New York Structural Biology Center, United States
Abstract
PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site.
Data availability
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Structure of the PP2C Phosphatase Domain and a Fragment of the Regulatory Domain of the Cell Fate Determinant SpoIIE from Bacillus SubtilisPublicly available at the RCSB Protein Data Bank (accession no: 5UCG).
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Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilis in a crystal form without domain swappingPublicly available at the RCSB Protein Data Bank (accession no: 5MQH).
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Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilisPublicly available at the RCSB Protein Data Bank (accession no: 3T91).
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CRYSTAL STRUCTURE OF THE HSLUV PROTEASE-CHAPERONE COMPLEXPublicly available at the RCSB Protein Data Bank (accession no: 1G3I).
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CRYSTAL STRUCTURE OF THE H. INFLUENZAE PROTEASE HSLV AT 1.9 A RESOLUTIONPublicly available at the RCSB Protein Data Bank (accession no: 1G3K).
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Crystal structure of RsbX in complex with manganese in space group P21Publicly available at the RCSB Protein Data Bank (accession no: 3W43).
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Structure of Orthorhombic crystal form of Pseudomonas aeruginosa RssBPublicly available at the RCSB Protein Data Bank (accession no: 3F7A).
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Crystal structure of pyruvate dehydrogenase phosphatase 1 (PDP1)Publicly available at the RCSB Protein Data Bank (accession no: 2PNQ).
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Structure of a C.elegans sex determining proteinPublicly available at the RCSB Protein Data Bank (accession no: 4JND).
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Crystal structure of SnRK2.6 in complex with HAB1Publicly available at the RCSB Protein Data Bank (accession no: 3UJG).
Article and author information
Author details
Funding
National Institutes of Health (GM18568)
- Richard Losick
Wellcome (82829)
- Anthony J Wilkinson
Damon Runyon Cancer Research Foundation (DRG 2051-10)
- Niels Bradshaw
Jane Coffin Childs Memorial Fund for Medical Research
- Christina M Zimanyi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Bradshaw et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases
eLife 6:e26111.
https://doi.org/10.7554/eLife.26111
Further reading
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N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
