A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases
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
-
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).
-
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).
-
Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilisPublicly available at the RCSB Protein Data Bank (accession no: 3T91).
-
CRYSTAL STRUCTURE OF THE HSLUV PROTEASE-CHAPERONE COMPLEXPublicly available at the RCSB Protein Data Bank (accession no: 1G3I).
-
CRYSTAL STRUCTURE OF THE H. INFLUENZAE PROTEASE HSLV AT 1.9 A RESOLUTIONPublicly available at the RCSB Protein Data Bank (accession no: 1G3K).
-
Crystal structure of RsbX in complex with manganese in space group P21Publicly available at the RCSB Protein Data Bank (accession no: 3W43).
-
Structure of Orthorhombic crystal form of Pseudomonas aeruginosa RssBPublicly available at the RCSB Protein Data Bank (accession no: 3F7A).
-
Crystal structure of pyruvate dehydrogenase phosphatase 1 (PDP1)Publicly available at the RCSB Protein Data Bank (accession no: 2PNQ).
-
Structure of a C.elegans sex determining proteinPublicly available at the RCSB Protein Data Bank (accession no: 4JND).
-
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.
Metrics
-
- 3,005
- views
-
- 547
- downloads
-
- 34
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We have previously shown that Disp promotes proteolytic solubilization of Shh from its lipidated terminal peptide anchors. This process, termed shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as a soluble sink for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is necessary and sufficient for Disp-mediated transfer because artificially cholesteroylated mCherry associates with HDL in a Disp-dependent manner, whereas an N-palmitoylated Shh variant lacking C-cholesterol does not. Disp-mediated Shh transfer to HDL is completed by proteolytic processing of the palmitoylated N-terminal membrane anchor. In contrast to dual-processed soluble Shh with moderate bioactivity, HDL-associated N-processed Shh is highly bioactive. We propose that the purpose of generating different soluble forms of Shh from the dual-lipidated precursor is to tune cellular responses in a tissue-type and time-specific manner.
-
- Biochemistry and Chemical Biology
- Cell Biology
Troubleshooting is an important part of experimental research, but graduate students rarely receive formal training in this skill. In this article, we describe an initiative called Pipettes and Problem Solving that we developed to teach troubleshooting skills to graduate students at the University of Texas at Austin. An experienced researcher presents details of a hypothetical experiment that has produced unexpected results, and students have to propose new experiments that will help identify the source of the problem. We also provide slides and other resources that can be used to facilitate problem solving and teach troubleshooting skills at other institutions.