To regulate the activity of proteins, cells often modify them by adding or removing chemical groups called phosphates. Therefore, the enzymes that add or remove these phosphate groups must be tightly regulated so that they are active at the right time and place. Enzymes known as phosphatases remove phosphate groups from proteins and the PP2Cs are one such family of enzymes that are found in bacteria, plants and animals. Despite their broad importance, it was not clear how cells control the PP2Cs.

One way to understand how an enzyme is controlled is to compare the three-dimensional structures of the enzyme when it is active and when it is inactive. Bradshaw et al. used a PP2C enzyme from bacteria as a model to understand how the cell regulates other PP2Cs.

The experiments reveal that the bacterial enzyme has a structural element that acts as a switch to control its activity. The phosphatase needs to bind metal ions to be active, and movement of the switch promotes binding of the metal ions to activate the phosphatase. The switch is also found in other members of the PP2C family. Furthermore, members of a seemingly unrelated family of enzymes called the proteasomal proteases, which degrade proteins, also have a similar architecture and are controlled by a similar switch. Thus, the phosphatase and protease families may have a common evolutionary history.

Multiple members of the PP2C family are involved in cancer and other diseases. The discovery of a regulatory switch provides new opportunities to use drugs to control phosphatase activity in patients. Many cancer drugs that are currently in use or are under development target enzymes that add phosphate groups to proteins, but efforts to target the phosphatases have largely been unsuccessful. Bradshaw et al.’s findings may enable the development of new drugs that target protein phosphatases.

Reversible protein phosphorylation is widely used in biological systems to control the activity of enzymes or the association of proteins with other proteins. Kinases and phosphatases control the phosphorylation state of target proteins in response to specific cellular or environmental cues, making reversible phosphorylation a flexible mechanism to control diverse biological systems (Huse and Kuriyan, 2002; Shi, 2009; Taylor and Kornev, 2011). Here we address the question of how members of the PP2C family of serine/threonine phosphatases are regulated to control processes such as cell growth and death, development, and responses to stress in all kingdoms of life (Kerk et al., 2015; Lammers and Lavi, 2007; Shi, 2009). Among serine/threonine phosphatases, a distinctive feature of the PP2C family is that the activity of a conserved catalytic domain is controlled by diverse regulatory domains that are often linked in cis to the catalytic domain (Shi, 2009; Zhang and Shi, 2004). We investigated the PP2C family member SpoIIE, which controls the activation of the cell-specific transcription factor σ^F during the developmental process of sporulation in the bacterium Bacillus subtilis.

Sporulation involves the formation of an asymmetrically-positioned septum that divides the developing cell into large and small cellular compartments (Stragier and Losick, 1996). SpoIIE is the most upstream member of a three-protein pathway that activates σ^F in the small cell (Figure 1A). It does so by dephosphorylating the phosphoprotein SpoIIAA-P (Duncan et al., 1995). Dephosphorylated SpoIIAA, in turn, displaces σ^F from the anti-sigma factor SpoIIAB to release the free and active transcription factor (Figure 1A) (Diederich et al., 1994). A long-standing mystery is how SpoIIE is regulated to generate dephosphorylated SpoIIAA selectively in the small cell. Recent work indicates that SpoIIE initially associates with the asymmetrically-positioned cytokinetic ring and then during cytokinesis is handed off to the adjacent cell pole, which will become the small cell (Bradshaw and Losick, 2015). Cell-specific activation is mediated by the self-association of SpoIIE molecules in the small cell, which protects the protein from proteolysis and activates the phosphatase (Bradshaw and Losick, 2015). Here we focus on the molecular mechanism of phosphatase activation.

Figure 1 with 2 supplements see all

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Like other PP2C family phosphatases, the catalytic center of SpoIIE uses two divalent cations (manganese in the case of SpoIIE) to deprotonate a water molecule that serves as the nucleophile for dephosphorylation (Arigoni et al., 1996; Schroeter et al., 1999). This active site is embedded in the conserved fold of the PP2C domain, which is shared by all PP2C family members (Shi, 2009). The PP2C domain is paired with diverse regulatory modules (over 1500 unique domain architectures have been identified in the InterPro database) (Mitchell et al., 2015), but how these regulatory modules control phosphatase activity was not understood. Here we identify a pair of α-helices at the heart of the regulatory mechanism that rotate to position a carbonyl oxygen to bind an active site Mn²⁺ ion and activate SpoIIE. We present evidence that this mechanism is widely conserved among PP2C family members. Remarkably, rotation of equivalent α-helices is also used to control an unrelated catalytic mechanism in the structurally similar family of enzymes that form the catalytic core of the proteasome (Arciniega et al., 2014; Ruschak and Kay, 2012; Shi and Kay, 2014; Sousa et al., 2000). This raises the possibility that PP2C phosphatases and proteasome proteases have a common evolutionary history that is linked by a shared regulatory mechanism.

We have presented the structures of the active and inactive state of the PP2C phosphatase SpoIIE from B. subtilis. Based on these structures, analysis of the function of SpoIIE mutants in vivo, and biochemical experiments, we propose that the movement of two helices at the base of the phosphatase domain, forming the switch region, activates the phosphatase by positioning the carbonyl oxygen of a conserved glycine to coordinate manganese in the active site. Importantly, and as we will explain, structural and functional data additionally suggest that the switch mechanism is broadly conserved among PP2C phosphatases. Unexpectedly, and underscoring the flexibility and conservation of the switch, our analysis also reveals that a similar module controls the activity of proteases that form the catalytic core of the proteasome. This raises the possibility that the switch helices are a shared, and possibly evolutionarily conserved, feature of at least two families of enzymes that use unrelated catalytic mechanisms.

The SpoIIE regulatory switch is broadly conserved among PP2C phosphatases

The following illustrative examples highlight the conservation and adaptability of the allosteric regulatory mechanism among PP2C phosphatases (Figure 5):

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RsbP

The phosphatase RsbP from B. subtilis is activated in response to energy stress by binding to its partner (RsbQ) to activate the transcription factor σ^B (Vijay et al., 2000) (Figure 5—figure supplement 1A). Gain-of-function mutants of RsbP that constitutively activate σ^B in the absence of RsbQ identified two elements in RsbP that control PP2C phosphatase activity (Brody et al., 2009). One element corresponds to one of the two switch helices we identified for SpoIIE (α1). The other element (designated as α0 by Brody et al.) was from the RsbP regulatory domain, and comparison with the structure of a closely related phosphatase (Levchenko et al., 2009) (RssB; the structure of RsbP itself has not been solved) suggests that this helix contacts the switch (Figure 5—figure supplement 1B). This suggests that RsbP and related phosphatases use the α0 helix as a regulatory module to position the switch to control phosphatase activity. This supports the broad conservation of the switch mechanism and suggests that the switch is controlled by docking varied input domains with the switch helices.

Pdp1

Pyruvate dehydrogenase phosphatase (Pdp1) dephosphorylates pyruvate dehydrogenase to promote respiratory metabolism (Vassylyev and Symersky, 2007) (Figure 5—figure supplement 2A). Human Pdp1 activity is inhibited by phosphorylation at a site distant from the active site (Y94), and phosphorylation at Y94 is commonly observed in human cancer cells and contributes to the Warburg effect (Shan et al., 2014). In the Pdp1 structure Y94 contacts a structural motif unique to Pdp1 (Vassylyev and Symersky, 2007) that packs against a pair of α-helices structurally homologous to the SpoIIE switch helices (Figure 5—figure supplement 2B). We hypothesize that phosphorylation of Y94 would displace this structural element, shifting the position of the switch helices and inhibiting Pdp1 activity through a mechanism similar to that for SpoIIE regulation. Additionally, Pdp1 is activated by binding to the lipoyl moiety on the E2 subunit of the pyruvate dehydrogenase complex, and the proposed lipoic acid binding site is contained in the same structural element that is contacted by Y94 and packs against the switch helices (Vassylyev and Symersky, 2007) (Figure 5—figure supplement 2B). Thus, our model for PP2C regulation may explain how Pdp1 integrates both positive and negative regulatory signals to control phosphatase activity.

Fem-2

The C. elegans Fem-2 phosphatase regulates sex determination in complex with its regulatory partners Fem-1 and Fem-3 (Chin-Sang and Spence, 1996) (Figure 5—figure supplement 3A). Additionally, the mammalian homologue of Fem-2 promotes caspase-dependent apoptosis by antagonizing Ca²⁺/calmodulin-dependent protein kinase (Tan et al., 2001). Fem-2 has a specific N-terminal regulatory domain that is the scaffold for binding Fem-1 and Fem-3 to form the active complex (Zhang et al., 2013). In the Fem-2 structure this regulatory domain packs against the equivalent of the switch helices (Zhang et al., 2013) (Figure 5—figure supplement 3B). How Fem-2 phosphatase activity is regulated is not clear, but the direct contact between the Fem-2 regulatory domain and the switch helices is consistent with our proposed model for regulation of phosphatase activity through the switch helices.

Hab1

The PP2C phosphatase Hab1 is a member of a sub-family of related phosphatases that regulate drought tolerance in response to abscisic acid in plants (Ma et al., 2009; Park et al., 2009) (Figure 5—figure supplement 4A). It is the only PP2C phosphatase for which a structure bound to its protein substrate (the kinase SnRK2) is available (Soon et al., 2012). Hab1 contacts SnRK2 primarily through a sub-domain (termed the ‘flap’ [Pullen et al., 2004]) that is variable in PP2C phosphatases and that packs against the switch helices (blue in Figure 5—figure supplement 4B and C) (Soon et al., 2012). In the presence of abscisic acid, the PYR/PYL/RCAR family of abscisic-acid-binding proteins inhibit Hab1 by binding to a site that overlaps with the binding site for SnRK2, suggesting that the switch could be influenced both by substrate and regulator binding. This could be a more general feature of PP2C phosphatases; the corresponding substrate-binding domain in SpoIIE changed conformation upon SpoIIE dimerization and activation (Figure 5—figure supplement 4D), suggesting that the conformational change of the switch could couple regulatory inputs to substrate binding. Additionally, coupling between substrate binding and the active conformation of the switch helices would provide a conserved mechanism to achieve the known high substrate specificity of PP2C phosphatases.

RssB

RssB activates the general stress response in E. coli, P. aeruginosa, and certain other gamma proteobacteria (Battesti et al., 2011). Although RssB is closely related to PP2C phosphatases, its primary role is not as a phosphatase (and does not require phosphatase activity), but rather as an adapter protein, delivering the transcription factor σ^S for degradation by ClpXP in the absence of stress (Figure 5—figure supplement 5A). Structural and genetic studies revealed that adapter activity is regulated by contacts between the RssB regulatory domain and the switch helices that are mediated by dimerization, similar to our observations for SpoIIE (Battesti et al., 2013; Levchenko et al., 2009) (Figure 5—figure supplement 5B and C). Because the primary function of an adapter protein is to mediate protein-protein interactions, we hypothesize that for RssB the switch couples regulatory inputs to substrate binding (rather than to phosphatase activity) through a mechanism such as proposed above for Hab1. Thus, the switch mechanism may not only provide a flexible platform for adapting phosphatase activity to various inputs but also to control different outputs. We note that there are other pseudo-PP2C phosphatases including Tab1, which mediates caspase dependent apoptosis (Lu et al., 2007) and which may have similarly repurposed the switch mechanism.

Based on these examples, we conclude that the SpoIIE regulatory switch is broadly used to control diverse PP2C phosphatases via regulatory domains that dock on the switch to couple phosphatase activity to regulatory inputs (Figure 5).

The PP2C switch is shared with the proteasome proteases

One of the most striking discoveries of our investigation is that the PP2C regulatory switch strongly resembles the allosteric switch that regulates the family of proteases that form the catalytic core of the proteasome (Arciniega et al., 2014; Ruschak and Kay, 2012; Shi and Kay, 2014; Sousa et al., 2000). These proteases are the most structurally similar family to PP2C phosphatases as revealed using the DALI server (Holm and Rosenström, 2010) and the ECOD database (Cheng et al., 2014), and like PP2C phosphatases their catalytic activity is subject to allosteric regulation. Specifically, the proteasome proteases and PP2C phosphatases have a conserved core fold (Figure 6A and B), which includes the switch helices, and the active sites are positioned in the same overall part of the structure. Although the proteases use different functional groups to mediate catalysis, the carbonyl oxygen of a conserved glycine (G629 of SpoIIE) at the junction of the core domain and the switch helices is used by both enzyme families for catalytic activity (Sousa et al., 2000).

Figure 6

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Association with the regulatory cap activates the proteases, ensuring that the proteolytic active sites are sequestered prior to activation (Seol et al., 1997) (Figure 6C). Early studies on HslV, the E. coli homologue of the proteasome proteases, revealed that allosteric activation by the HslU cap takes place by rotation of the switch helices to position the active site glycine (Figure 6D) (Sousa et al., 2000). This mechanism is remarkably similar to the regulatory mechanism we proposed for PP2C phosphatases; docking of a regulatory module repositions the structurally homologous region in the same way to position the same functional group to achieve catalytic activity (Video 2).

Video 2

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This mechanism is also conserved for the archaeal proteasome, which like the eukaryotic proteasome includes an additional layer of related, but catalytically inactive α subunits; docking of the cap displaces the switch helices of the α subunits, which directly contact and reposition the switch helices of the catalytic β subunits (Ruschak and Kay, 2012). Several lines of evidence suggest that this mechanism is conserved for the eukaryotic proteasome (Arciniega et al., 2014) and is additionally used by chaperones that promote proteasome maturation (Wani et al., 2015). Finally, comparative studies of the constitutive proteasome and the immune proteasome suggested that differences in the conformational flexibility of the switch underlies their differences in activity (Arciniega et al., 2014).

Thus, our identification of the PP2C switch demonstrates that PP2C phosphatases and the proteasome use the same allosteric regulatory module, revealing an unexpected link between two fundamental signaling systems – reversible phosphorylation and regulated proteolysis. Independent analysis of structural and sequence similarity suggest that this is a result of common evolutionary ancestry. Structural comparison by the ECOD database, which classifies the evolutionary relationships of protein folds places PP2C phosphatases and the proteasome proteases in the same ‘X-group’, which is consistent with homology (Cheng et al., 2014). Independently, sequence-based searches using HHPRED (Söding et al., 2005) detected weak sequence similarity between phosphatases and the broad family of NTN-hydrolases that includes the proteasome proteases. For example, using the SpoIIE phosphatase domain sequence to search hidden Markov model alignments for B. subtilis proteins identified weak sequence similarity to D-fructose-6-phosphate amidotransferase, an NTN hydrolase. Notably, the region of possible sequence similarity maps to the switch helices and the β strands that follow and pack with the switch (although it is not known whether the switch helices play a regulatory role in amidotransferases).

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Author details

1. Niels Bradshaw

   Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

   Contribution

   NB, Conceptualization, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-6845-4717

2. Vladimir M Levdikov

   Structural Biology Laboratory, Department of Chemistry, University of York, York, United Kingdom

   Contribution

   VML, Conceptualization, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Christina M Zimanyi

   Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

   Present address

   New York Structural Biology Center, New York, United States

   Contribution

   CMZ, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-6782-507X

4. Rachelle Gaudet

   Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

   Contribution

   RG, Supervision, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-9177-054X

5. Anthony J Wilkinson

   Structural Biology Laboratory, Department of Chemistry, University of York, York, United Kingdom

   Contribution

   AJW, Conceptualization, Funding acquisition, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4577-9479

6. Richard Losick

   Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States

   Contribution

   RL, Conceptualization, Funding acquisition, Writing—original draft, Writing—review and editing

   For correspondence

   losick@mcb.harvard.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-5130-6582

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