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Targeting the Arginine Phosphatase YwlE with a Catalytic RedoxBased Inhibitor Jakob Fuhrmann,† Venkataraman Subramanian,† and Paul R. Thompson*,†,‡ †
Department of Chemistry and ‡The Kellogg School of Science and Technology, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States S Supporting Information *
ABSTRACT: Protein phosphatases are critical regulators of cellular signaling in both eukaryotes and prokaryotes. The majority of protein phosphatases dephosphorylate phosphoserine/phosphothreonine or phosphotyrosine residues. Recently, however, YwlE, a member of the low-molecular weight protein tyrosine phosphatase (LMW-PTP) family, was shown to efficiently target phosphoarginine. YwlE shares several sequence motifs with this family including the C(X)4 CR(S/T) motif that is crucial for catalysis and redox regulation of the enzyme. Herein we confirm that Cys9 and Cys14 play important roles in YwlE catalysis and regulation. On the basis of these observations, we designed and synthesized a YwlE inhibitor, denoted cyc-SeCN-amidine, that irreversibly inhibits YwlE (kinact/KI = 310 M−1 min−1) by inducing disulfide bond formation between the two active site cysteine residues. Interestingly, inactivation appears to be catalytic, since the compound is neither destroyed nor altered after enzyme inhibition. Although the exact mechanism of disulfide induction remains elusive, we propose several potential mechanisms accounting for the cyc-SeCNamidine mediated inhibition of YwlE. These findings could stimulate the design of similar selenium-based compounds targeting other redox-sensitive enzymes.
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steric bulk but also alters electrostatic interactions; the net charge of a phosphoarginine is −1. Arginine phosphorylation is particularly well suited to disrupt protein−DNA interactions that rely on the positive charge of the guanidinium group of arginine, as in the case of the transcriptional repressor CtsR, which is tightly bound to specific DNA promoter elements but loses its affinity for DNA upon arginine phosphorylation.8 Based on sequence analyses, YwlE belongs to the LMW-PTP family (Supplementary Figure 1), which contains a conserved active site signature motif, C(X)4CR(S/T), that forms the base of the active site cleft.9,13 The dephosphorylation mechanism occurs in a two-step process and involves the formation of a covalent phosphothioester reaction intermediate that is generated by nucleophilic attack of the active site cysteine on the incoming phosphoarginine residue. Studies with other LMW-PTPs, where the active site cysteines possess low pKa values (typically ∼514,15), suggest that the active site cysteine in YwlE, Cys9, exists as the negatively charged thiolate anion (R− S−) at physiological pH. In this form, the cysteine residue acts as a strong nucleophile. However, in this state it is also particularly vulnerable to oxidation via reactive oxygen species (ROS; e.g., hydrogen peroxide). Given the high sensitivity to ROS, it is unsurprising that these enzymes have adapted mechanisms to resist ROS-mediated irreversible inactivation,
rotein phosphatases regulate a wide variety of cellular signaling events, modulate several metabolic pathways, and are crucial for maintaining overall cellular physiology.1−3 These processes are mediated by several classes of protein phosphatases targeting distinct phosphoresidues. Members of the PPP and PPM phosphatase family dephosphorylate phosphoserine/threonine residues, whereas phosphotyrosine residues are targeted by the versatile protein tyrosine phosphatase (PTP) family.4 In addition, SixA, a member of the histidine phosphatase superfamily efficiently hydrolyzes the phosphoramidate bond of phosphohistidine.5 Recently a novel class of protein phosphatase was identified that can release the phosphate group from phosphoarginine residues.6,7 Protein arginine phosphorylation represents a novel posttranslational modification (PTM) that alters protein function in vitro and in vivo.6,8 For example, the protein arginine kinase McsB catalyzes the selective phosphorylation of proteinembedded arginine residues, and the protein arginine phosphatase YwlE reverses this reaction (Figure 1A). Based on a proteomic study, more than 100 phosphoarginine sites could be mapped in Bacillus subtilis.6 Furthermore, the B. subtilis strain lacking YwlE exhibits impaired resistance to ethanol stress.9 Numerous cellular pathways, including the stress response, protein degradation, cell motility, and competence, are affected by arginine phosphorylation.6 Physiological effects on such a diverse range of cellular processes are conceivable because arginine residues play key roles in protein−protein, protein−DNA, and protein−RNA interactions,10−12 and arginine phosphorylation not only adds © 2013 American Chemical Society
Received: February 27, 2013 Accepted: June 21, 2013 Published: July 9, 2013 2024
dx.doi.org/10.1021/cb4001469 | ACS Chem. Biol. 2013, 8, 2024−2032
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Figure 1. Regulation of protein arginine phosphorylation and dephosphorylation. (A) The protein arginine kinase McsB phosphorylates the guanidinium group of arginine. The reverse reaction is catalyzed by YwlE. The phosphomoiety attached to the guanidinium group of the arginine substrate is highlighted in red. (B) Structural model of YwlE highlighting critical cysteine residues. In addition to the cysteine residues, the structural model illustrates critical active site residues, including Asp118, which is involved in acid−base catalysis; Arg15, which is required for the proper alignment of the phosphate moiety; and Phe120, which may be required to generate a deep active site pocket. YwlE from B. subtilis (PDB code: 1ZGG) was used as the structural template for homology modeling using Modeler software. The figure of the model was produced using PyMOL (9v10) software.
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RESULTS AND DISCUSSION Mutational Analysis of YwlEs Active Site Cysteine Residues. Given the homology between YwlE and other LMW-PTPs (Supplementary Figure 1), we focused our initial efforts on developing irreversible inhibitors for this enzyme because we hypothesized that such compounds would react with either Cys9, the active site cysteine, or Cys14, an auxiliary cysteine residue present in the active site (Figure 1B). As a first step toward this goal, we set out to confirm the predicted roles of Cys9 and Cys14. As stated above, Cys9 is thought to play a direct role in substrate dephosphorylation by forming the initial phosphothioester adduct. In contrast, the function of Cys14 is less clear, but the equivalent residue in bovine LMW-PTP, which is located in the active site cleft on the phosphate binding P-loop, has been suggested to activate an incoming water molecule and promote the hydrolysis of the phospho-enzyme intermediate.21 Additionally, this residue is appropriately positioned to prevent the ROS-mediated irreversible inactivation of the enzyme by forming a disulfide adduct with Cys9; based on the NMR solution structure of YwlE derived from B. subtilis, the distance between the corresponding thiols of Cys9 and Cys14 is ∼4 Å.22 To evaluate the contribution of Cys9 and Cys14 to YwlE catalysis, we mutated these residues to serine and analyzed the effects of these mutations on catalytic activity (Table 1). Briefly, YwlE C9S was devoid of any significant detectable phosphatase activity (kcat/KM ≤ 2 × 10−4 M−1 s−1), consistent with its role as the essential nucleophile in the dephosphorylation reaction. In contrast, the YwlE C14S mutant is still active, albeit with a significant reduction in kcat (kcat = 0.19 ± 0.01 s−1 versus 1.12 ± 0.01 s−1 for the wild type enzyme); there was essentially no effect on KM. These results are consistent with Cys9 being the
and it is now clear that these processes have evolved to reversibly regulate phosphatase activity in response to a variety of cellular signals.16−18 For example, the bovine LMW-PTP enzyme contains a second cysteine residue in close proximity to the catalytic cysteine, and its role is to prevent the irreversible oxidation of the thiolate to sulfinic and sulfonic acid by forming an intramolecular disulfide bond upon oxidation to sulfenic acid; the disulfide is readily reduced by glutathione or thioredoxin.19 A variety of other phosphatases, e.g., PTEN, CDC25C, KAP, and MKP3, use a similar protective mechanism. In contrast to this mechanism, the tyrosine phosphatase PTP1B is protected from further oxidation by the formation of a covalent sulfenyl-amide between the active site cysteine residue and the neighboring backbone amide group.20 Although numerous YwlE substrates have been putatively identified, the physiological roles of this enzyme and how it is regulated are poorly defined. Therefore, we set out to develop YwlE inhibitors that could be used as chemical probes to analyze the function of YwlE. Toward that goal, we describe herein the generation of the first irreversible YwlE inhibitor, cyc-SeCN-amidine, which inactivates YwlE (kinact/KI = 310 M−1 min−1) via the reversible oxidation of its critical active site cysteine residues. Interestingly, inactivation appears to be catalytic because the compound is neither destroyed nor altered after enzyme inhibition. Although the exact mechanism of disulfide bond formation remains elusive, several potential mechanisms are proposed. In addition to providing insights into the regulation of this enzyme, our findings will likely stimulate the design of similar selenium-based compounds targeting other redox-sensitive enzymes. 2025
dx.doi.org/10.1021/cb4001469 | ACS Chem. Biol. 2013, 8, 2024−2032
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and 13C NMR analysis, including NMR correlation methods (e.g., COSY and HSQC), excluded the presence of the linear configuration and point to the presence of the intramolecular cyclized isomers 4a and 4b containing a 2-imino-dihydroselenazol and 2,4-diiminoselenazolidine ring structure respectively (Scheme 1). These molecules, which are not distinguishable by mass analysis and are generated in a 3:1 ratio (4a:4b), likely arise by nucleophilic attack of either one of the two amidine nitrogen atoms onto the electrophilic SeCN carbon. Although the structural isomers 4a and 4b could not be separated by HPLC, we did confirm the presence of two selenium containing compounds by 77Se NMR analysis (Supplementary Figure 2). Hereafter, the mixture of isomers is denoted cyc-SeCN-amidine To initially examine the inhibitory properties of cyc-SeCNamidine, we followed product formation as a function of time in the absence and presence of increasing amounts of inhibitor. With inhibitor, the progress curves are curved (Figure 2A), consistent with time-dependent inhibition of the enzyme. To verify that cyc-SeCN-amidine is in fact an irreversible inhibitor, the preformed enzyme·cyc-SeCN-amidine complex was dialyzed overnight, and the residual activity was determined using our standard YwlE activity assay. The results of these experiments indicate that there is no recovery of activity, consistent with cyc-SeCN-amidine being an irreversible YwlE inhibitor (Figure 2B). From the data in Figure 2A, it was possible to extract values for the pseudo first-order rate constants for enzyme inactivation. Plots of kobs versus inhibitor concentration are linear (Figure 2C), suggesting that cycSeCN-amidine acts as a quiescent affinity label. From this analysis, the kinact/KI value is 310 M−1 min−1 (Table 2), making this compound the most potent YwlE inhibitor described to date. Mechanism of Inactivation. To begin to understand the mechanism by which cyc-SeCN-amidine inhibits YwlE, we initially used ESI-MS to measure the intact mass of YwlE that had been treated with and without the compound. The untreated sample revealed a major peak at 17,233 Da that closely matched the expected theoretical mass of 17,233.5 Da (Figure 3A). In contrast, the inhibitor-treated sample has a mass that is 2 Da lower, that is, 17,231 Da, than predicted for
Table 1. Steady State Kinetic Parameters of Different YwlE Variants
a
enzyme
KM (mM)
kcat (s−1)
kcat/KM (M−1 s−1)
YwlE YwlE C9S YwlE C14S
100 ± 10 NDa 115 ± 14
1.12 ± 0.01 NDa 0.19 ± 0.01
11.1 ≤2 × 10−4 1.69
ND, not determined.
active site nucleophile and Cys14 playing a role in substrate turnover. Identification of cyc-SeCN-Amidine As a Potent YwlE Inhibitor. Having demonstrated that Cys9 and Cys14 are critical for catalysis, we set out to generate an irreversible inhibitor for YwlE. Given our experience in developing inhibitors for other arginine modifying enzymes, including the protein arginine deiminases (PADs) and protein arginine methyltransferases (PRMTs),23−27 we first tested whether Clamidine (compound 1 in Scheme 1) could inhibit YwlE because the reactive haloacetamidine moiety present in this molecule reacts with a variety of different arginine modifying enzymes including the PADs, agmatine deiminases, and the PRMTs.23−27 However, Cl-amidine proved to be a rather poor YwlE inhibitor (kinact/KI ≤ 1.1 M−1 min−1), likely because the distance between the active site nucleophile and the reactive center on Cl-amidine is too great. Given this finding, we considered that the replacement of the chlorine atom with a selenocyanate would effectively decrease the distance between the active site cysteine and the reactive moiety. We expected that Cys9 would attack the selenium atom and displace the cyano group, thereby forming a covalent adduct between YwlE with compound 3 in Scheme 1. This compound, which is denoted SeCN-amidine, was readily accessed on the solid phase by reaction of the resin bound Cl-amidine intermediate with KSeCN in DMF (Scheme 1); Cl-amidine was synthesized on Knorr-amide resin according to an established method.28 SeCN-amidine was obtained in good yield after cleavage from the resin with TFA and purification by reverse phase HPLC. Although the target structure was the linear form of SeCNamidine (compound 3), detailed one- and two-dimensional 1H
Scheme 1. Solid-Phase Synthesis of cyc-SeCN-Amidine Starting from Cl-Amidinea
a
The synthesis of SeCN-amidine resulted in the cyclized isomers depicted on the right. The SeCN group is highlighted in blue. The relative stoichiometry of compounds 4a and 4b is ∼3:1 as shown by 77Se NMR analysis (Supplementary Figure 2). 2026
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Figure 2. cyc-SeCN-amidine mediated inactivation of YwlE. (A) Progress curve of YwlE inactivation by the indicated concentrations of cyc-SeCNamidine. (B) YwlE preincubated with cyc-SeCN-amidine and then dialyzed overnight in the absence of DTT does not show any recovery of phosphatase activity. (C) Rate of inactivation, kobs, versus inhibitor concentration. (D) YwlE preincubated with cyc-SeCN-amidine followed by overnight dialysis and treatment with DTT showed substantial recovery of phosphatase activity.
Table 2. Inhibition Constants of YwlE Treated with cycSeCN-Amidine and H2O2 kinact/KI (M‑1 min‑1)
a
compound
− catalase
+ catalase
cyc-SeCN-amidine H2O2
310 ± 18 3930 ± 70
302 ± 12 NIa
NI, no inhibition at 10 mM.
the wild type unmodified enzyme (Figure 3B). The formation of either a disulfide bond or sulfenylamide would account for this 2 Da mass decrease. Since YwlE only contains two cysteine residues (i.e., Cys9 and Cys14) and these residues are present in close proximity within the active site and are either critical (Cys9) or important (Cys14) for catalysis (see above), the simplest interpretation is that cyc-SeCN-amidine inactivates YwlE by inducing the formation of an intramolecular disulfide bond between Cys9 and Cys14. To verify that inactivation is due to formation of a disulfide bond, the cyc-SeCN-amidine inactivated enzyme was digested with trypsin and then subjected to LC−MS/MS analysis to map the site of modification (Supplementary Figure 3). Analysis of the YwlE tryptic digest yielded a single peptide (ILFVCTGNTCR) encompassing a loss of 2 Da compared to its theoretical mass. This peptide was selected for further fragmentation by CID. While all observed y ions (C-terminal fragments) contained a mass shift of −2 Da, all b ions (Nterminal fragments) except b10 , showed the predicted
Figure 3. ESI-MS analysis of YwlE incubated with cyc-SeCN-amidine. ESI-mass analysis of the deconvoluted spectra of YwlE (A) and YwlE treated with cyc-SeCN-amidine (B).
unmodified mass; the b10 ion contains a mass shift of −2 Da. In total, these data indicate that the modification must be 2027
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Figure 4. Potential mechanisms of cyc-SeCN-amidine (compound 4a) mediated inactivation of YwlE (see text for details).
located between Cys9 and Cys14. In addition, we could not detect any b or y ions resulting from fragmentation between both cysteine residues. These fragments would generate the same mass as the precursor ion due to covalent linkage of Cys9 and Cys14. These data unequivocally proved the presence of an intramolecular disulfide bond between Cys9 and Cys14. To further confirm disulfide bond formation, we preformed the enzyme·inhibitor complex, dialyzed the protein and then incubated the enzyme in the absence and presence of DTT; DTT should reduce the hypothesized disulfide bond. As shown
in Figure 2B, cyc-SeCN-amidine treated YwlE shows no recovery of activity after overnight dialysis, unless DTT is added to the buffer (Figure 2D). Given that cyc-SeCN-amidine induces disulfide bond formation, we were curious to determine the fate of the compound after enzyme inactivation. For these studies, we incubated cyc-SeCN-amidine with stoichiometric amounts of wild type YwlE. We then removed the enzyme using a low molecular weight desalting column and analyzed the residual cyc-SeCN-amidine sample by LC−MS. Surprisingly, there was 2028
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not shown). These data indicate that cyc-SeCN-amidine is not a nonspecific ROS generator. To further explore the redox-based inactivation of YwlE, we performed ESI-MS analysis on enzyme treated with increasing concentrations of cyc-SeCN-amidine. H2O2 treated YwlE was used as a control. The results of these studies revealed that cycSeCN-amidine only generates the disulfide species even at millimolar concentrations of the compound. In contrast, YwlE is highly resistant to being irreversibly inactivated, similarly to other PTPs, because high concentrations of H2O2 are required to generate the sulfinic (HSO2) and sulfonic acid (HSO3) modified forms of the enzyme; at lower concentrations the intramolecular disulfide form predominates (Supplementary Figure 8). These results also demonstrate that cycSeCN is not a nonspecific generator of ROS. Having ruled out Mechanism 1, we explored the validity of the remaining three redox-based mechanisms (Figure 4B−D). Initially we examined the susceptibility of cyc-SeCN-amidine to oxidation but were unable to detect an oxidized form of cyc-SeCN-amidine (see Mechanism 2), even after treatment with 100 mM H2O2 (not shown). This result is inconsistent with Mechanism 2. Since the above results are difficult to reconcile with Mechanisms 1 and 2, we reasoned that it might be possible to use the YwlE C14S mutant to trap the covalent intermediates predicted by Mechanisms 3 and 4 (Figure 4C and D). For these studies the YwlE C14S mutant was treated with cyc-SeCN-amidine, and the presence of an adduct was detected by MS (Figure 5). As expected, incubation of cycSeCN-amidine with the YwlE C14S mutant (17,217 Da) did not result in the loss of 2 Da (indicative of disulfide bond formation). Instead, the major species detected corresponds to a covalent enzyme·cyc-SeCN-amidine inhibitor complex (17,597 Da) (Figure 5B); the two additional minor species correspond to different oxidation states of the enzyme (indicated by the presence of the plus 32 and 48 Da species). The 380 Da mass increase (−1H + cyc-SeCN-amidine) is consistent with the adducts depicted in Mechanisms 3 and 4. Note that as a control, we also treated the YwlE-C14S mutant with H2O2 and detected only the formation of multiple oxidized species (Figure 5C). In addition, by demonstrating that it is possible to generate a covalent adduct to cyc-SeCNamidine, these data further rule out Mechanism 1. To both verify adduct formation and determine the fate of cyc-SeCN-amidine, the C14S mutant was first treated with cycSeCN-amidine, and then we selectively removed unbound cycSeCN-amidine, either by Ni-NTA purification of the Histagged enzyme or by desalting using 2 kDa cutoff membranes. Residual covalently bound inhibitor was then eluted from the protein by the addition of DTT. The isolated inhibitor was then analyzed by measuring the resulting eluted inhibitor fraction by LC−MS. The results of these studies revealed that there was no change in the mass of cyc-SeCN-amidine (Supplementary Figure 9). Importantly, the isotope distribution pattern observed for the cyc-SeCN-amidine compound matches the theoretical selenium isotope distributions, further confirming the presence of the intact molecule. In summary, these data confirm the existence of the covalent adduct and demonstrate that upon release from the enzyme, the molecule is regenerated to the intact cyc-SeCN-amidine. Since these data imply that cyc-SeCN-amidine is regenerated during the cycle of YwlE inactivation, we first treated YwlE with inhibitor and then isolated it and retested its ability to inactivate fresh YwlE. As a control, we also isolated cyc-SeCN-amidine
no difference in the mass or isotope distribution pattern of cycSeCN-amidine before and after enzyme treatment (Supplementary Figure 4). Thus cyc-SeCN-amidine induces disulfide bond formation but remains intact. Potential Modes of Inactivation. Given the above findings, at least four potential inactivation mechanisms can be drawn (Figure 4 and Supplementary Figure 5). Since we do not know the identity of the reactive species, Figure 4 and Supplementary Figure 5 depict the potential inactivation mechanisms afforded by 4a (the major isomer) and 4b (the minor isomer), respectively. First, the formation of a noncovalent complex between cyc-SeCN-amidine and preoxidized YwlE could induce a conformational change in the active site to promote intramolecular disulfide bond formation (Figure 4A). Alternatively, oxidation of the selenium atom in the cyc-SeCNamidine warhead (Figure 4B), either before enzyme binding or within the active site of the enzyme, generates an electrophile that reacts with the active site thiolate to form a covalent enzyme-cycSeCN-adduct stabilized by a S−Se bond. The cycSeCN-amidine moiety is almost immediately displaced by the attack of the neighboring cysteine residue forming the more stable S−S bond and regenerating the compound (Figure 4B). This mechanism is analogous to the one used by methionine sulfoxide reductase.29 A third possible mechanism involves nucleophilic attack of Cys9 at an electrophilic carbon on the selenazol ring, which results in the generation of a covalent adduct that is resolved by disulfide bond formation with Cys14 (Figure 4C). The transient buildup of negative charge on the ring is almost immediately transferred to molecular oxygen to generate a peroxoselenazol intermediate that ultimately collapses to form H2O2, analogous to the reoxidation mechanism used by flavin peroxidases. Finally, the fourth mechanism, which also involves attack on the same carbon of the selenazol ring, generates a tetrahedral intermediate that collapses to generate the ring opened species. The covalent adduct is then resolved as above, leading to the formation of negatively charged intermediate that reacts with the oxidized selenium atom to regenerate the warhead (Figure 4D). To begin to differentiate between these potential modes of inactivation, we first examined the inhibitory properties of H2O2. For these studies, product formation was followed as a function of time in the absence and presence of increasing H2O2 concentrations. The results of this analysis indicate that H2O2 is a highly potent YwlE inactivator (kinact/KI = 3930 ± 70 M−1 min−1; see Table 2 and Supplementary Figure 6B). In fact, it is ∼10-fold more potent than cyc-SeCN-amidine and like cycSeCN-amidine readily reversed by the addition of DTT (Supplementary Figure 7). This result is inconsistent with Mechanism 1 because if cyc-SeCN-amidine simply causes a conformational change that promotes intramolecular disulfide bond formation with pre-existing oxidized enzyme, then one would expect H2O2 to be a significantly worse inhibitor than cyc-SeCN-amidine. To explore the possibility that cyc-SeCN-amidine effects enzyme inactivation by acting as a ROS generator, we examined the effect of catalase and superoxide dismutase (SOD) on the inhibitor properties of this compound. The effect of catalase on H2O2 mediated enzyme inactivation was used as a control. As shown in Table 2 and Supplementary Figure 6, catalase completely blocks the effect of H2O2, whereas this enzyme does not diminish the potency of cyc-SeCN-amidine; SOD had no effect on cyc-SeCN-amidine induced enzyme inactivation (data 2029
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formation is facile, which is inconsistent with the existence of preoxidized enzyme and the notion that cyc-SeCN-amidine could induce a conformational change that promotes enzyme inactivation via this route. Although Mechanism 2 includes a covalent adduct of the correct mass, and is precedented for methionine sulfoxide reductase,29 this mechanism is inconsistent with the fact that we are unable to detect the oxidized form of cyc-SeCN-amidine, even when this compound is treated with 100 mM of H2O2. Although, we cannot detect any oxidized cyc-SeCN-amidine, it remains formally possible that cyc-SeCN-amidine is oxidized within the unique environment of the active site to facilitate enzyme inactivation via this mechanism. Although the two remaining routes satisfy the above criteria, they do include the rather unsatisfactory need to invoke the existence of carbanion-like intermediates for the reoxidation of the selenazol ring. Additionally, Mechanism 4 requires that the selenium atom be oxidized, via an unknown mechanism, so as to facilitate the ring-closure reaction. Given our observations thus far, and the mechanistic similarity to flavin peroxidases, Mechanism 3 is the most satisfying. However, we have been unable to verify both the requirement for oxygen as the ultimate electron acceptor in the reaction and the formation of the resulting peroxide byproduct. As such, Mechanism 4 is most consistent with our results and will serve as a model to further refine our understanding of this highly unique YwlE inactivator in the future. Regardless of the specific mechanism of inactivation, it is clear that the active form of cyc-SeCN-amidine is readily regenerated during the cycle of enzyme inactivation, suggesting that this compound irreversibly inactivates the enzyme by acting catalytically as an electron shunt between the cysteine thiols on the enzyme and an ultimate electron acceptor. Given the ease of synthesis of cyc-SeCN-amidine and the efficient conversion of active YwlE to its inactive oxidized form, the cycSeCN-amidine warhead will likely provide a unique approach for promoting the facile oxidative inactivation of phosphatases and other enzymes and proteins containing two vicinal cysteine residues. To test this hypothesis we examined the effect of cycSeCN-amidine on a related bacterial tyrosine phosphatase, YfkJ, which also belongs to the LMW-PTP family and contains two adjacent cysteine residues in the active site (embedded within the C(X)4CRS/T motif). Cyc-SeCN-amidine inhibits YfkJ with a similar degree of potency to YwlE (kinact/KI = 665 ± 10 M−1 min−1; Supplementary Figure 10A), indicating that cyc-SeCNamidine also targets other phosphatases that contain two vicinal cysteine residues in their active site. cyc-SeCN-amidine also induces a disulfide bridge formation in YfkJ, as observed by intact mass analysis (Supplementary Figure 10B). To further characterize the specificity of cyc-SeCN-amidine, we incubated varying amounts of this compound with E. coli cell extracts. To detect free protein sulfhydryl groups, we labeled proteomes with rhodamine-iodoacetamide. As can be seen in Supplementary Figure 11, YwlE is efficiently modified by cyc-SeCNamidine, indicated by a diminished rhodamine signal, while the labeling efficiency of other protein bands is differentially affected. These data indicate that cyc-SeCN-amidine possesses some selectivity toward particular protein thiols. Given the above data, we expect that this warhead will serve as a unique chemical tool that can be used to characterize the reactivity of the thiol proteome, similar to other cysteine-reactive electrophilic probes.
Figure 5. ESI-MS analysis of YwlE C14S incubated with cyc-SeCNamidine and H2O2. ESI-mass spectra of YwlE C14S (A), YwlE C14S treated with cyc-SeCN-amidine (B), and YwlE C14S treated with H2O2 (C).
that had been treated identically, except in the absence of enzyme. The results of these experiments indicate that the kincat/KI of the isolated YwlE treated compound (kincat/KI = 180 ± 35) is virtually identical to that obtained for the control (kincat/KI = 325 ± 30), thus demonstrating that cyc-SeCNamidine is regenerated after it is released from the enzyme. Conclusions. Given the above results, it is clear that cycSeCN-amidine inactivates YwlE by inducing disulfide bond formation between the two vicinal active site cysteines, that is, Cys9 and Cys14. The mechanism of inactivation, however, is less clear. Nevertheless, any mechanism must satisfy the following criteria: (1) cyc-SeCN-amidine initially forms a covalent adduct with Cys9 that is then converted to the disulfide linked species. (2) The reduced cyc-SeCN-amidine, which is formed upon disulfide bond formation, is reoxidized to regenerate the starting material. Given our results with the YwlECys14 mutant, where DTT treatment releases the intact compound, this oxidation step appears to occur nonenzymatically. (3) cyc-SeCN-amidine does not inactivate the enzyme through the nonspecific generation of H2O2 or O2−, as catalase and SOD do not decrease the rates of inactivation. The only mechanisms that satisfy all three criteria are Mechanisms 3 and 4. As described above, Mechanism 1 is ruled out by the fact that it does not include a covalent adduct. Additionally, H2O2 is a better YwlE inactivator than cyc-SeCNamidine, thereby indicating that once oxidized, disulfide bond 2030
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ESI Mass Spectrometry Measurements. Wild type and mutant YwlE samples were desalted prior to nanoESI MS analysis using C18 ZipTips (Millipore). Salts were removed by washing with 0.1% formic acid (FA) in water, and subsequently the bound proteins were eluted with 0.1% FA in acetonitrile. Desalted proteins were directly infused into the LTQ mass spectrometer (Thermo-Fisher Scientific) using static nanoelectrospray conditions. Proteins were analyzed in the positive ion mode. Mass-to-charge ratios were extracted from the raw data, deconvoluted, and deisotoped with the MaqTran software. MS/MS Analysis To Identify the Site of Modification. To determine the site modified by cyc-SeCN-amidine, inactivated YwlE was separated by SDS-PAGE, the corresponding band was excised from the gel and digested with trypsin overnight, and the resulting peptide mixture was analyzed using LC−ESI-MS/MS on a LTQ mass spectrometer. Theoretical digest masses were calculated using MSProduct from the Protein Prospector proteomics web tools. Chemical Synthesis of cyc-SeCN-Amidine. Knorr Amide MBHA (Novabiochem) resin (200 mg, 0.186 mmol) was treated twice with 7 mL of 20% piperidine in dimethylformamide (DMF) with gentle rocking at rt for 10 min each. Then, the resin was washed three times with 7 mL of DMF. Fmoc-Orn(Dde)-OH (289 mg, 0.558 mmol) was coupled to the resin using O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (211 mg, 0.558 mmol) and hydroxybenzotriazole (HOBt) (75.4 mg, 0.558 mmol) dissolved in 7 mL of DMF. N-Methylmorpholine (NMM) (0.124 mL, 1.116 mmol) was added, and the reaction allowed to proceed at rt for 5 h. The resin was collected by filtration and washed with DMF (3 × 7 mL). Fmoc removal after the coupling step was effected with 20% piperidine in DMF (2 × 7 mL) for 20 min, collected by filtration, and washed with DMF (3 × 7 mL). To attach the benzoyl group, benzoylchloride (0.0872 mL, 0.744 mmol) and NMM (0.124 mL, 1.116 mmol) were mixed in DMF (7 mL) and allowed to react at rt. The suspension was rocked gently for 3 h, and the resin was collected by filtration and washed with DMF (3 × 7 mL). Removal of the Dde protecting group was accomplished by treating the resin twice with 2% hydrazine (in 7 mL DMF) for 1 h each. The resin was collected by filtration and washed 3 times with 7 mL of DMF. Ethyl chloroacetimidate(HCl) (116.8 mg, 0.744 mmol) and triethylamine (0.1572 mL, 1.116 mmol) were added, and the suspension was rocked gently overnight. The SeCN group was attached by addition of 26.8 mg (0.186 mmol) of KSeCN in 7 mL of DMF, followed by washing with 3 × 7 mL DMF. The resin was collected by filtration, washed with DMF (3 × 7 mL) and DCM (3 × 7 mL), and dried under vacuum. The dried resin was treated with a mixture (2 × 8 mL, 1 h each) of TFA/DCM (70/30). The cleavage solutions were collected by filtration, combined, and concentrated under a stream of nitrogen. The resulting compound was purified by semipreparative reverse phase HPLC using a water/methanol gradient to afford the product as an offwhite hygroscopic powder. The identity and purity of the resulting compound was validated by mass spectrometry on a quadrupole LC− MS (Agilent) mass spectrometer (expected mass for C15H19N5O2Se (M + H)+: 382.07 Da, observed mass: 382.10 Da). Compound 4a: 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.93−7.88 (m, 2H), 7.58−7.44 (m, 3H) 4.63 (s, 2H), 4.41 (dt, 1H), 3.56 (q, 2H), 1.94−1.53 (m, 4H). 13 C NMR (DMSO-d6, 100 MHz) δ (ppm): 183.66, 180.74, 173.44, 166.34, 134.04, 131.30, 128.15, 127.47, 52.68, 45.71, 34.63, 28.83, 25.20. 77Se NMR (DMSO-d6, 76.3 MHz) δ (ppm): 419.9 (t). Compound 4b: 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 7.93−7.88 (m, 2H), 7.58−7.44 (m, 3H) 4.67 (s, 2H), 4.41 (dt, 1H), 1.94−1.53 (m, 4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 185.40, 182.67, 173.44, 166.34, 134.04, 131.30, 128.15, 127.47, 52.68, 48.43, 35.72, 28.83, 25.14. 77Se NMR (DMSO-d6, 76.3 MHz) δ (ppm): 393.8 (t).
METHODS
Site Directed Mutagenesis. Full length YwlE phosphatase from Bacillus stearothermophilus containing a C-terminal hexa-histidine tag was used for functional analyses. All expression constructs were derived from a pET21a(+) expression vector (Stratagene). The following constructs were generated by site directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Stratagene). To generate the YwlE C14S mutant, the gene coding for YwlE derived from B. stearothermophilus was mutated using the oligonucleotide 5′gggctccggcttgtattgcccgtgcaaacg-3′ and its reverse complement. The mutation coding for YwlE C9S was introduced using oligonucleotide 5′-tattgcccgtgctaacgaacaaaatgcgatatggc-3′ and its reverse complement. All constructs were verified by DNA sequence analysis. Protein Expression and Purification. The YwlE phosphatase constructs were overexpressed in Escherichia coli BL21 (DE3) cells. Cells were grown at 37 °C in LB medium in the presence of ampicillin (100 μg/mL), and expression was induced with 0.5 mM IPTG for 2 h at an OD600 nm of 0.8. After centrifugation at 7,000 rpm, the cell pellet was resuspended in 300 mM NaCl, 50 mM Tris/HCl pH 8.0 and disrupted by sonication. The cleared lysate was loaded on a Ni2+nitrilotriacetate (Ni-NTA) column (Qiagen), and the His-tagged proteins were eluted by applying a stepwise imidazole gradient at 250 mM imidazole. The phosphatase-containing fraction was directly applied to a Superdex-75 column (prep grade, GE Healthcare) equilibrated with 20 mM Tris/HCl pH 7.6, 100 mM NaCl. Fractions containing YwlE were pooled and concentrated using a Centricon concentrator (Millipore) with a 10 kDa nominal molecular mass cutoff. The concentrated protein was flash frozen in liquid nitrogen and stored at −80 °C YwlE Phosphatase Activity Assay. The phosphatase activity of YwlE was tested using p-nitrophenylphosphate (pNPP) as the substrate. The dephosphorylation assay was performed in 20 mM Tris/HCl pH 7.6 using 50 mM substrate. The reaction was initiated by addition of 0.5 μM phosphatase followed by incubation in 96-well microplates (BD) at 25 °C. The phosphatase reaction was monitored in 20 s intervals at 405 nm (absorption maximum of the generated pnitrophenolate, pNP) using a SpectraMax M5 (Molecular Devices) microplate reader. Reaction times, amounts of enzyme, and concentrations of substrates were optimized to have linear kinetics. The concentration of the generated pNP was calculated using a pNP standard curve. For kinetic studies, the initial rates obtained were fit to eq 1
v = Vmax[S]/(K m + [S])
(1)
using GraFit version 5.0.11. To evaluate the inhibitory properties of the analyzed compounds, progress curves were generated. For these experiments, inhibitors were added in assay buffer containing 50 mM pNPP. The values for kinact/KI were obtained by multiplying the apparent kobs.app values by the transformation 1 + [S]/Km to obtain the pseudo first-order rate constant, kobs, and these values were plotted versus inhibitor concentrations and fit to eq 2
kobs = k inact[I]/(K i + [I])
(2)
using GraFit version 5.0.11. kinact is the maximal rate of inactivation; KI is the concentration of inactivator that yields half-maximal inactivation, and [I] is the concentration of inactivator. All kinetic experiments were performed at least in duplicate, and inter-replicate errors were typically less than 20%. Dialysis Experiments. To test whether cyc-SeCN-amidine is an irreversible YwlE inactivator, cyc-SeCN-amidine was preincubated with the phosphatase for 1 h and then dialyzed overnight against 20 mM Tris-HCl (pH 7.6) and 100 mM NaCl. The resulting activity present in these samples was quantified using the assay described above and compared to that of a control reaction mixture that was treated identically without addition of cyc-SeCN-amidine. To confirm the reversibility of inactivation, DTT (final concentration 10 mM) was added to the reaction mixture.
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ASSOCIATED CONTENT
S Supporting Information *
Supplementary Figures 1−11 including detailed figure legends. This material is available free of charge via the Internet at http://pubs.acs.org. 2031
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ACS Chemical Biology
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by The Scripps Research Institute (to P.R.T.) and by an EMBO Long Term Fellowship (to J.F.).
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ABBREVIATIONS LMW-PTP, low molecular weight-protein tyrosine phosphatase; SeCN, selenocyanate; DTT, dithiothreitol; ESI-MS, electrospray ionization-mass spectrometry; LC−MS, liquid chromatography−mass spectrometry
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REFERENCES
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