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A glutamate switch controls voltage-sensitive phosphatase function

Ciona intestinalis voltage sensing phosphatase Ci-VSP couples a voltage-sensing domain (VSD) to a lipid phosphatase similar to the tumor suppressor PTEN. How the VSD controls enzyme function has been unclear. Here, we present high-resolution crystal structures of the Ci-VSP enzymatic domain that reveal conformational changes in a key loop, termed the “gating loop”, that controls access to the active site by a mechanism in which residue Glu411 directly competes with substrate. Structure-based mutations that restrict gating loop conformation impair catalytic function and demonstrate that Glu411 also contributes to substrate selectivity. Structure-guided mutations further define an interaction between the gating loop and linker that connects the phosphatase to the VSD for voltage control of enzyme activity. Together, the data suggest that functional coupling between the gating loop and the linker forms the heart of the regulatory mechanism that controls voltage-dependent enzyme activation.


Introduction
Ci-VSP, the Ciona intestinalis voltage-sensing phosphatase, is the founding member of a novel membrane protein family in which a transmembrane voltage-sensing domain (VSD) controls activity of a C-terminal cytoplasmic enzyme domain that has sequence similarity to the tumor suppressor PTEN (protein lipid phosphatase and tensin homologue deleted on chromosome 10) rather than an ion channel pore [1][2][3] . This VSD-PTEN protein class is found in frogs, fish, mice, and humans 4 and provides a direct means to couple membrane potential changes with phosphatase action 1,5,6 . Despite extensive biophysical characterization, structural underpinnings of how voltage controls Ci-VSP function have remained unclear. Two distinct conceptual models have been forwarded to explain how the VSD response to voltage changes drives enzymatic activity of the cytoplasmic domain: simple movement of active site to the membrane 6 and a voltage-dependent conformational change in the active site 7 .
To define elements that influence Ci-VSP function, we determined six Ci-VSP cytoplasmic domain high-resolution X-ray crystal structures of different conformations and variants and one substrate analog complex. These reveal an enzymatic domain loop, the "gating loop", that differs substantially from its PTEN counterpart, the "TI loop", and that displays three distinct conformations that open or close active site access by controlling the position of gating loop residue Glu411. Functional investigation of a battery of structure-based mutants evaluated in the isolated enzymatic domain in vitro and full-length protein in live cells show that Glu411 contributes to activity. Further, our data suggest that the linker region that connects the VSD to the enzymatic domain and is known to be important for function 6,7 , couples voltage-sensing to enzyme activity by controlling gating loop action. Together, the results provide a new model in which gating loop conformational changes play an essential role in how voltage regulates Ci-VSP activity.
The Ci-VSP 241 Form I structure revealed three unanticipated differences from PTEN that impact the active site (Fig. 1c). (1) Similar to Matsuda et al. 13 , the C2 domain β-strand 6-7 loop (Ci-VSP residues 512-526, the "522 loop") is in a different orientation than the corresponding PTEN loop (PTEN residues 259-270, the "CBR3 loop") and contributes Tyr522 to the active site (Fig. 1c, d). (2) The phosphatase domain loop between helices 5 and 6 (residues 398-413, the "gating loop"), which is five residues longer than the homologous TI loop in PTEN (PTEN residues 159-169), adopts a conformation, differing from the lower-resolution Ci-VSP structure 13 , that buries Glu411 inside a hydrophobic pocket comprising Gly371, Leu391, Phe394, and Ile416, from α-helices 4, 5, and 6, denoted the "α456 pocket" (Fig. 1c-e). (3) The linker region (residues 241-263) folds as an α-helix followed by a β-hairpin (Fig. 1b, c). The α-helix, absent from previous Ci-VSP structures 13 , coincides with the expected C-terminal end of the transmembrane S4 segment and positions residues important for coupling voltage-sensing to activity 6,7 , Lys252 and Arg253, near the gating loop and active site, respectively. Differences in gating loop and linker conformations between 241 Form I and the structures of PTEN and a prior Ci-VSP construct 13 suggest that these segments may be mobile and have roles in function.

Multiple Ci-VSP structures reveal gating loop movement
The core regions of the structures of two other 241 C363S crystal forms, Forms II and III, ( Table 1, Supplementary Fig. 1b, c) and a C363S construct having a truncated linker domain, denoted "256" (residues 256-576, C363S) ( Table 1, Supplementary Fig. 1d) were similar to 241 Form I (Fig. 2a, b, Supplementary Table 1). However, the linker and gating loop had notable differences. The extent of α-helical structure in the linker varied in the three 241 structures whereas the β-hairpin remained. In 256, the visible part of the linker common to 241 and 256 (residues 258-263) had no regular secondary structure (Fig. 2b). The gating loop conformations in 241 Form I and 241 Form III were similar, whereas this region adopted a different conformation in the 241 Form II and 256 structures (Fig. 2c, d).
Further comparison revealed that even though the gating loop N-terminal end showed a range of conformations ( Fig. 2a, b), some of which make minor crystal lattice interactions (Supplementary Table 2), a four-residue C-terminal segment (residues 409-412) adopted just two (Fig. 2c, d) that placed Glu411 in either the hydrophobic α456 pocket (241 Forms I and III) (Fig. 2c) or the active site (241 Form II, both chains, and 256) (Fig. 2c, d). This second conformation, similar to that seen by Matsuda et al. 13 , docks Val410 rather than Glu411 in the α456 pocket (Fig. 2c, d). Having Glu411 in the α456 pocket leaves the active site wideopen ("open") ( Fig. 2e), whereas having Glu411 in the active site ("closed") leaves only a small pocket occupied by a sulfate ion in the 256 structure (Fig. 2f) and should preclude substrate binding. Refinement of 241 Form I revealed a minor conformation in which Glu411 has a similar position to that in 256. The reduced active site pocket size in the closed form is striking given prior evidence that a 256 construct bearing a catalytically competent active site is inactive 7 . This, together with the observation of open and closed conformations, suggests that the conserved gating loop 409-412 segment ( Supplementary  Fig. 3) forms a switch controlling active site access and catalytic domain activity.

Active site architecture
The Ci-VSP P-loop signature motif (residues His362-Arg369) adopts the canonical structure of protein tyrosine phosphatases 11,14 (Supplementary Figure 2a, b) and PTEN 15 (Fig. 1c, d) and is exceptionally well ordered, having B-factors among the lowest in the structure (Supplementary Fig. 2c-e). The active site pocket is lined by a loop that contributes general acid-base residues for catalysis, (residues 330-333, the "D331 loop") in a conformation identical to PTEN (Fig. 1d). The D331 loop is joined by Tyr522, which closes off a shallow pocket occupied by solvent in PTEN (Fig. 1d, Supplementary Fig. 2f) and whose hydroxyl is oriented in a manner seemingly ideal for substrate interactions. The 241 Form I electron density also revealed a phosphate ion coordinated by Arg369, the P-loop element commonly used to bind substrate phosphates 11 (Fig. 1d). This phosphate position coincides with a sulfate ion in 256 and substrate phosphate in PTP1B phosphatase-substrate complexes 8,16 ( Supplementary Fig. 2b). Thus, it likely marks the position of the phosphate that Ci-VSP would cleave from its substrates.
Residues central to the active site, Ser363-Gly366, adopt two distinct conformations (  Table 3). Nevertheless, this active site mobility may influence catalysis. Together, the multiple structures point to active site and gating loop conformational changes that we set out to investigate in turn.

Roles of Gly365 and Tyr522 in enzymatic activity
Ci-VSP cleaves the 5-phosphate of PI(3,4,5)P 3 and PI(4,5)P 2 17-19 and has weaker activity against the 3-phosphate 17 . In contrast, PTEN acts solely on the PI(3,4,5)P 3 3phosphate 17,20,21 . These specificity differences have been suggested to originate from an Ala→Gly difference at Ci-VSP active site residue 365 4,17 ; however, the 241 Form I structure shows that Ci-VSP and PTEN active sites are identical (RMSD = 0.29 Å and 0.77 Å, C α and all atoms, respectively) ( Fig. 1d, Supplementary Table 1). To investigate whether the Ala→Gly difference might affect substrate specificity, we conducted a series of functional assays on full-length Ci-VSP G365A in live Xenopus oocyte membranes in the context of a second mutation, G214C, that serves as a fluorescent label attachment site at the S4 transmembrane segment external end to assess membrane expression and VSD conformational changes 5 .
To test specificity further, we used a set of green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains that bind to different phosphoinositides: a PI(4,5)P 2 probe, phospholipase C (PLC) PH domain (GFP-PLC-PH) 18,19 (Fig. 4b); a PI(3,4,5)P 3 probe, general receptor for phosphoinositides-1 (GRP1) PH domain (GFP-GRP-PH) (Fig.  4c); and a PI(3,4)P 2 probe, the tandem PH domain-containing protein 1 (TAPP-1) PH domain (GFP-TAPP-PH) 19 (Fig. 4d). For PI(4,5)P 2 and PI(3,4,5)P 3 probes (Fig. 4b, c), following depolarization, both WT and G365A, but not C363S, caused similar probe mobilization, albeit with different magnitudes, suggesting that WT and G365A have similar substrate selectivity. For the PI(3,4)P 2 probe (Fig. 4d), WT caused a fluorescence increase that was followed by a decrease. These changes correspond to the probe moving to the membrane because of PI(3,4)P 2 generation from PI(3,4,5)P 3 , and then away from the membrane, indicating PI(3,4)P 2 destruction. In contrast, only a small fluorescence decrease was observed with G365A, indicating preservation of some 3-phosphatase activity against PI(3,4)P 2 . Consistent with prior work 17 , all assays showed that G365A has reduced activity relative to WT as indicated by smaller ΔF amplitudes. This reduction is consistent with the idea that active site flexibility is important and is restricted by the introduction of the sidechain methyl group.
To test whether Tyr522 influences Ci-VSP function, we examined the Y522A mutant. Such behavior suggests that Y522A slows dephosphorylation at the PI(3,4)P 2 3-phosphate.
We used a malachite green assay 1,7,17 to measure activity of purified WT and Y522A 241 cytoplasmic domain constructs against PI(3,4,5)P 3 and PI(3,4)P 2 . Circular dichroism showed that Y522A does not cause a loss of structure ( Supplementary Fig. 4c). Y522A modestly reduced steady-state activity against PI(3,4)P 2 relative to WT, but without a significant effect on V max ( Fig Table 3), indicating no major change to the isolated enzyme fragment substrate specificity. However, Y522A accelerated VSD activation and deactivation rearrangements in voltage-clamp fluorometry (VCF) measurements of tetramethyl-rhodamine-maleimide (TMRM) labeled full-length protein (Fig. 4f). This is reminiscent of how other catalytic site mutations affect VSD motion 6,7,18 and together with the effects on GFP-probe behavior supports the idea that Tyr522 has a role in Ci-VSP function.

Gating loop conformation and Ci-VSP function
To address the hypothesis that gating loop mobility plays a role in controlling enzyme activity, we made a set of Glu411 hydrophobic substitutions (E411L, I, and F) that should favor residue 411 occupancy of the hydrophobic α456 pocket and stabilize the open conformation. We also made a deletion (Δ401-405) that removed gating loop residues having the greatest structural variation to create a Ci-VSP gating loop of equal length to the PTEN TI loop (Fig. 5a).
Malachite green assays showed that all three hydrophobic mutants reduced PI(3,4,5)P 3 dephosphorylation ( Fig. 5b) with E411F causing the biggest reduction. Δ401-405 resulted in a near complete loss of activity (Fig. 5b). All three Glu411 hydrophobic mutants and Δ401-405 eliminated activity of full-length Ci-VSP measured in oocytes using the Kir2.1Q reporter assay (Fig. 5c). In contrast, polar substitutions (E411D, Q, and T) had a milder impact on the isolated enzymatic domain and full-length protein (Fig. 5b, c).
We determined the structures of the two gating loop mutants that caused the most dramatic functional losses, E411F and Δ401-405 (  Table 3).
Notably, neither gating loop conformation matches the equivalent PTEN region in which Thr167, the 411 counterpart, is midway between open and closed conformations (Fig. 5g). This, together with the functional data showing that E411F (open) and Δ401-405 (closed) dramatically impair activity of the isolated domain and full-length protein (Fig. 5b, c), suggests that the structures represent trapped forms of two key 410-412 segment conformations. The structures further suggest that gating loop movement, centered on the exchange of residues 410 and 411 in the α456 hydrophobic pocket, is part of the Ci-VSP catalytic cycle.
To probe the gating loop further, we examined E411T and E411F in the full-length protein expressed in Xenopus oocytes using the GFP-PH domain assay and VCF. E411F did not generate detectable PI(3,4)P 2 (Fig. 5h). In contrast, E411T caused an unexpected increase in GFP-TAPP-PH membrane fluorescence that lacked the subsequent late declining phase seen with WT (Fig. 5h). This was accompanied by a slowing of the fluorescence increase (Supplementary Figure 4a, b), suggesting that E411T moderately slows PI(3,4,5)P 3 5phosphate dephosphorylation and strongly decreases PI(3,4)P 2 3-phosphate dephosphorylation. Kinetic studies on the isolated cytosolic domain confirmed the decrease in 3-phosphate dephosphorylation, showing a two-fold reduction in V max for the E411T cytosolic domain relative to WT (Supplementary Figure 4d, Supplementary Table 4). VCF measurements showed that both E411T and E411F altered VSD movement (Fig. 5i). E411T shifted the activation curve to the left (-13.2 mV), and slowed deactivation, suggesting active state stabilization. Conversely, E411F shifted the activation curve to the right (+11.3 mV) and slightly accelerated deactivation, consistent with resting state stabilization. Together, the sensitivity to changes at Glu411 supports the idea that gating loop motion is crucial to Ci-VSP function and suggests that Glu411 may have a role in substrate recognition.

A gating loop conformation similar to PTEN
The structure of a complex of a soluble substrate mimic, myo-inositol-1,4,5-trisphosphate (IP 3 ) and the 241 C363S construct (Table 1) revealed an IP 3 molecule in the active site ( Fig.  6a, b) and a gating loop in which Glu411 is between the open and closed positions (Fig. 6c).
Here, Val410 remains in the α456 pocket (Fig. 6d) and the active site conformation identical to 241 Form I (Supplementary Table 3). The active site IP 3 clashes sterically with the Glu411 closed conformation (Fig. 6b-d) and provides direct evidence that the gating loop must move to accommodate substrate binding. The remainder of the gating loop is very similar to the closed 241 Form II chain B. Strikingly, the Val410 and Glu411 conformations are exceptionally similar to the equivalent PTEN positions, including alignment of Ci-VSP Glu411 and PTEN Thr167 across the Cα-Cγ positions (Fig. 6d).
The IP 3 has its 1-phosphate at the position of the active site phosphate and sulfate from the 241 and 256 structures, respectively, (Fig. 6a). In native Ci-VSP inositol phosphate substrates, the 1-phosphate links the inositol ring to a lipid and thus, is unlikely to be cleaved. Nevertheless, a counterclockwise rotation of the IP 3 can be made to place the 5phosphate in the active site and 4-phosphate in a position where it could interact with Glu411.
The structure also revealed a second IP 3 bound to cationic residues, proximal to the active site, Arg281, Arg286, Arg313, and Lys364, (Supplementary Fig. 5). As this region is likely to appose the lipid bilayer, this second IP 3 site may represent a position for nonspecific interactions with the membrane or may have a more specific role in affecting substrate movement into or out of the active site.

Gating loop-linker coupling
To probe how voltage changes drive Ci-VSP function, we investigated the linker-gating loop relationship. Inspection of the N-terminal linker in 241 Form I, where this element is most completely defined, (Fig. 7a) suggested residues that might mediate gating loop-linker coupling: the conserved gating loop residues 398-400 ( Supplementary Fig. 3) and linker residues Lys252 and Arg253, known to couple voltage changes to activity 6,7 . In 241 Form I, gating loop Asp400 interacts via a salt bridge with Lys252 and linker residue Arg253 coordinates the Gly365 backbone carbonyl of the active site. Notably, Lys252 and Arg253, exchange positions between the 241 Form I and IP 3 bound structures (Fig. 7b). Lys252 goes from a solvent-exposed interaction with Asp400 to a position that makes a hydrogen bond to the Gly365 carbonyl. Conversely, Arg253 goes from coordinating the Gly365 carbonyl to a solvent exposed interaction with Asp400. This exchange is also present in the closed structure 241 Form II chain B (Supplementary movie). Gating loop Arg398 makes hydrogen bond interactions with the active site loop (Fig. 7a) in all of the structures. Based on the structures and evidence that K252Q and R253Q drastically reduce voltage-dependent activity and uncouple the VSD from the active site 7 , we changed Asp400 to Asn, Ala, Lys, and Arg. All were detrimental to enzymatic domain (Fig. 7c) and full-length protein function (Fig. 7d). None affected the structure as assessed by circular dichroism (Supplementary Fig.  6).
We next asked if Asp400 has a role in coupling the VSD and active site. Earlier analysis of this kind showed that linker residues Lys252 and Arg253 are critical for voltage dependent activity and for coupling catalytic site properties to the VSD 7 . Asp400 mutants that abolished enzymatic domain activity, D400K and D400R, almost eliminated the dramatic influence of a catalytic site mutation, D311A on VSD deactivation rate, consistent with disruption of VSD-active site coupling (Fig. 7e). Together, the data suggest that voltagedependent rearrangements of the VSD couple to the enzyme active site via interactions between linker residues Lys252 and Arg253 and their gating loop partner Asp400.

Discussion
Understanding how transmembrane voltage changes influence the activity of diverse membrane proteins remains a challenge 22 . In voltage-gated phosphatases two distinct models could explain how membrane depolarization regulates membrane lipid dephosphorylation. In one, analogous to the PTEN diffusion-based process where the Nterminal domain interacts with the membrane, depolarization would cause the Ci-VSP linker to interact with membrane phospholipids and place a constitutively active catalytic site within reach of substrate 6 . In a second model, depolarization would drive a conformational change that gates the catalytic activity 7 . Our studies strongly support the second.  (Fig. 5d, e). This arrangement should offer unfettered substrate access to the active site and yet, disrupts Ci-VSP function (Fig. 5b, c). This seeming paradox suggests that the gating loop does more than simply control active site access. Indeed, E411T influences both substrate selectivity and VSD movement (Fig. 5h,i). Thus, the data point to a mechanism in which the gating loop facilitates active site entry or exit of substrate, product, or both, and underscore a central role for Glu411 in this process.
Burial of a glutamate in a hydrophobic cavity, as seen with Glu411 in the α456 pocket, is unusual, but not unprecedented 23,24 . There are well-characterized examples where hydrophobic pockets interact with charged or polar ligands [25][26][27] . Nevertheless, such interactions have an energetic penalty that is likely to be an important component of gating loop function. Indeed, Glu411→hydrophobic mutations, which should partition into the α456 hydrophobic cavity, drastically reduce activity (Fig. 5b, c). This result fits with the concept of a tradeoff between stability and function for enzymatic residues involved in substrate binding and catalysis 28 and suggests that gating loop mobility depends on the hydrophilic character of residue 411.
Our findings also highlight the interdomain linker connecting the VSD and enzyme 1,6,7 . Structure-based mutational analysis indicates that two linker residues, Lys252 and Arg253, and gating loop residue Asp400 are key to linker-gating loop coupling. This is substantially different from the PTEN 29-31 membrane recruitment mechanism 6 and instead supports a mechanism driven by voltage-dependent conformational change in the active site 7 . Notably, the structural plasticity of the gating loop N-terminal end contrasts with the switch region conformational properties. Interactions of this portion of the gating loop with the lipid bilayer, absent from the structures, may be important for mediating VSD-switch region coupling.
The data presented here uncover a previously unknown element required for Ci-VSP function, the "gating loop". The gating loop switch region, residues 409-412, adopts three distinct conformations that we propose are part of the catalytic cycle (Fig. 7f), in which substrate is given active site access (open), occluded from the active site (closed), or selected (bound) by Glu411. The ability of the gating loop to access these conformations is controlled by the VSD and involves linker residues Lys252 and Arg253 and gating loop residue Asp400. Together, the results suggest that a mobile gate whose conformation is modulated by the linker that connects the cytoplasmic domain to the voltage-sensing S4 segment of the VSD mediates the voltage dependence of Ci-VSP enzyme activity.

Circular dichroism
Circular dichroism spectra were measured on an Aviv model 215 spectrometer (Aviv Biomedical) from 300 nm to 200 nm at 4°C using a 0.1 cm path length cuvette. Five scans of the same sample were averaged. Molar ellipticity was calculated as: [Θ]= 100(m)/Cnl, where m is the CD signal in millidegrees, C is protein concentration in millimolar, n is the number of residues, and l is path length in centimeters.  Diffraction data were collected at 100K from crystals flash frozen in liquid nitrogen, Beamline 8.3.1 (ALS, Berkeley, CA), by oscillation at λ= 1.116 Å with an ADSC Quantum 315r detector. For cryo-protection, crystals were passed through their corresponding well solution with addition of 18% glycerol for ammonium sulfate conditions or with PEG 2000 concentrations increased to 30% for PEG 2000 conditions. Data were processed using HKL2000 33 .

Structural determination and refinement
Structures were determined by molecular replacement using MolRep 34 and PTEN (PDB code: 1D5R 15 ) with non-conserved residues replaced by to alanine as a search model for the solution of 241 Form I and 256. 241 Form I and 256 structures were used for the other crystal forms, depending on the beginning residues. COOT 35 was used for graphical modeling. Structures were refined with Refmac 34 , and randomly selected 5% of data were used for R free . Typically, after the molecules were located in the asymmetric unit cell, rigidbody refinement was conducted at 3.5 Å (this refinement was re-performed later as needed), followed by restrained refinement. TLS refinement was applied. For resolution better than 1.5 Å, B factors were refined anisotropically; for the 241 Form I dataset, unrestrained refinement was performed at the end.

Molecular biology for full-length assays
Ci-VSP in the pSD64TF vector was provided by Y. Okamura (Osaka University). GFP-PLC-PH, GFP-TAPP-PH, GFP-GRP-PH were provided by T.

Electrophysiological measures of activity
Ci-VSP catalytic activity was measured by detecting Kir2.1 R228Q (Kir2.1Q) activation by PI(4,5)P 2 5 . The cell was hyperpolarized to −100 mV where Ci-VSP is off until a steady state current was established (5-7 minutes) followed by depolarization to +60 mV to activate Ci-VSP until a steady state was re-established (2-7 minutes). Resulting current changes between the Ci-VSP off and on states were measured and expressed as percent activity. Currents were leak subtracted by assuming a voltage-independent linear leak. Current was measured at −100 mV after the test holding potential, and leak was measured at +50 mV where the Kir2.1Q channels should be blocked. Leak-subtracted (LS) current was calculated as: I ls = I −100mV + 2I +50mV . Percent activity was calculated as ΔI/I max = (I +60mV first -I +60mVlast )/I +60mV first . 50 nl of mRNA was injected into X. laevis oocytes at a 40:1 to a 100:1 (μg μl −1 ) Ci-VSP:Kir2.1Q (total RNA ~0.9-2 μg μl −1

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.