Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity*

Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser36 as the major p70S6k phosphorylation site, along with a low frequency site at Thr40, using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 μmol/min/mg) of the activity of WT enzyme (95 μmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser36 is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser36 upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg104 in the adjacent subunit. Because Arg104 is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser36 inhibits the catalytic function of LTC4S by interference with the catalytic machinery.


Leukotriene C 4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C 4 (LTC 4 ). LTC
is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser 36 as the major p70S6k phosphorylation site, along with a low frequency site at Thr 40 , using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 mol/min/mg) of the activity of WT enzyme (95 mol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA 4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser 36 is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser 36 upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg 104 in the adjacent subunit. Because Arg 104 is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser 36 inhibits the catalytic function of LTC4S by interference with the catalytic machinery.
Leukotriene (LT) 2 C 4 synthase (LTC4S) catalyzes the formation of LTC 4 by conjugating the unstable allylic epoxide inter-mediate LTA 4 with reduced glutathione (GSH) (1). LTC 4 and its metabolites LTD 4 and LTE 4 are known as cysteinyl leukotrienes (cys-LTs), which are involved in bronchial asthma and allergic inflammatory disorders (1)(2)(3). The cys-LTs signal through two G-protein-coupled receptors, denoted CysLT1 and CysLT2, to exert their biological functions such as smooth muscle contraction and increased vascular permeability. Several drugs, typified by montelukast, have been developed that specifically target the CysLT1 receptor (4). Recently, additional G-protein-coupled receptors that recognize cys-LTs have been identified, in particular gpr17 and CysLT3 (5,6). The increasing complexity of cys-LT signaling has promoted research and drug development efforts targeting the upstream LTC4S as it catalyzes the committed step in cys-LT biosynthesis (7).
The leukotrienes are derived from arachidonic acid through the 5-lipoxygenase pathway where cytosolic phospholipase A 2 , 5-lipoxygenase, and 5-lipoxygenase-activating protein play important roles (7). Protein phosphorylation/dephosphorylation appear to be important regulatory mechanisms for cellular LT biosynthesis. The two key upstream enzymes, cytosolic phospholipase A 2 and 5-lipoxygenase, are regulated through phosphorylation events, apparently for activation (8) and translocation (9) to the nuclear membrane. LTC4S is yet another enzyme in the 5-lipoxygenase pathway that is regulated by intracellular phosphorylation (10,11).
LTC4S is an integral membrane protein that belongs to the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) superfamily whose six human members share structural similarity and form homotrimeric enzymes involved in arachidonic acid metabolism and detoxification (12). For 5-lipoxygenase-activating protein, LTC4S, and microsomal prostaglandin E synthase-1, crystal structures have been determined (13)(14)(15)(16). Besides LTC4S, no other MAPEG member has been reported to be regulated by phosphorylation. Phosphoregulation of LTC4S was first recognized when protein kinase C (PKC) activation of leukocytes was found to down-regulate LTC4S enzyme activity and attenuate cys-LT production (10). When LTC4S was cloned and sequenced, two putative PKC consensus sites (Ser-Ala-Arg) were found at positions 28 and 111 but were not investigated experimentally (17). In a recent study, it was demonstrated that a ribosomal S6 kinase (p70S6k) is responsible for the observed phosphoregulation of LTC4S in monocytes (18).
The aim of our study was to identify the site(s) on LTC4S that is phosphorylated by p70S6k and investigate the molecular mechanism for suppression of enzyme activity. We identified the sites with an in vitro phosphorylation assay, autoradiography, and mass spectrometry. The effects of phosphorylation on enzyme activity and kinetic properties were investigated using LTC4S with phosphorylation-mimicking mutations. To obtain a mechanistic explanation for our experimental results, we analyzed phosphorylated and unphosphorylated forms of LTC4S by comparative molecular dynamics (MD) and determined the crystal structure of the phosphomimetic S36E mutant.

Results and Discussion
Protein phosphorylation constitutes an essential part of the regulation of almost every aspect of cellular function (19). It has previously been shown that LTC4S is also regulated by phosphorylation, and p70S6k was found to be one of the key players in this process (10,18). The p70S6k is a serine/threonine-specific kinase localized both in the cytosol and nucleus (20). In this study, we show that the predominant p70S6k phosphorylation site on LTC4S is Ser 36 .
Prediction of Candidate Phosphorylation Site(s)-Initially, we used an online phosphorylation prediction tool, NetPhos 2.0 Server, to identify the potential phosphorylation site(s) based on sequence information using an artificial neural network method (21). The prediction identified six Ser, two Thr, and one Tyr residue (Fig. 1A). The major inclusion criteria for further characterization of individual residues were their predictive score and location in the structure. Thus, Ser 28 and Ser 36 had the highest probability scores of 0.810 and 0.987, respectively, whereas serine residues at positions 23, 57, 100, and 111 exhibited very low probability scores ranging from 0.002 to 0.159. Because Ser 23 , Ser 28 , Ser 57 , Tyr 97 , and Ser 111 are located within the membrane lipid bilayer (Fig. 1, B and C) with poor accessibility for kinases, we reasoned that the probability that these residues will be phosphorylated is very low. It is common that phosphorylation sites on membrane proteins are located in extramembrane loop regions as in human cardiac Na ϩ channel Na v 1.5 (22). In LTC4S, Ser 36 is located in a loop region, and Ser 100 is found just above the membrane-spanning region within the active site (Fig. 1C). The server also predicted two neighboring Thr residues at positions 40 and 41 with relatively high probability scores and located in the same loop as Ser 36 but with their side chains pointing toward the LTC4S trimer interface (Fig. 1C). It should also be noted that a serine/threoninespecific kinase was used for this study, and the relative abundance of the phosphorylated form of Ser, Thr, and Tyr has been reported as a ratio of 1800:200:1 in vertebrate cells (23). Hence, four of nine predicted residues were selected for further analysis, viz. Ser 36 , Ser 100 , Thr 40 , and Thr 41 .
Identification of Phosphorylation Site (s) on LTC4S by MS/MS Analysis-To identify the phosphorylation sites, in vitro phosphorylated proteins were analyzed by mass spectrometry. The MS analyses of LTC4S achieved up to 32% sequence coverage. The peptides containing Ser 36 , Thr 40 , Thr 41 , and Ser 111 could be identified in WT and S36A samples. The peptide covering Ser 28 , 3 DEVALLAAVTLLGVLLQAYFSLQVISAR 30 , was most likely too large and hydrophobic, whereas the peptide covering Ser 100 , 100 SAQLR 104 , was too short and hydrophilic. The peptides covering Ser 36 , Thr 40 , and Thr 41 , 35 VpSPPLTTGPPE-FER 48 and 32 AFRVpSPPLTTGPPEFER 48 where pS is phosphoserine, were repeatedly found to be phosphorylated. Only singly phosphorylated peptides were detected. In all but two cases, the peptides were phosphorylated at Ser 36 , and in the other two cases, peptides were phosphorylated at Thr 40 (Fig. 2), suggesting that Ser 36 is the predominant phosphorylation site. No evidence was found for phosphorylation of Thr 41 (supplemental Fig. S1).
To increase the chance of identifying phosphorylation at Ser 100 , two mutants were used in which the tryptic cleavage site immediately prior to or immediately after the Ser 100 had been mutated. The R99H was specifically generated for this study, whereas the mutation R104A was published previously (24). MS analyses identified 93 YFQGYAHSAQLR 104 and 100 SAQ-LALAPLYASAR 113 but only as the non-phosphorylated peptides. Hence, we could not find any mass spectrometric evidence for phosphorylation of Ser 100 .
Analysis of LTC4S Phosphorylation by Autoradiography-It has been shown previously that LTC4S can be phosphorylated FIGURE 1. Phosphorylation sites in LTC4S predicted with an online tool, NetPhos 2.0 Server. A, the probability scores for each predicted residue. B, the predicted sites are marked in the LTC4S primary structure with the membrane-spanning regions underlined in green. Predicted phosphorylation sites located within the membrane are marked in blue, and the four residues that were selected for further characterization are marked in red. C, predicted serine phosphorylation sites are indicated in the LTC4S trimeric structure (Protein Data Bank code 2UUH). DDM, a detergent molecule that is supposed to bind at the same site as LTA 4 , is indicated in red, and the second substrate GSH is shown in blue within the active site. Residues within the membrane-spanning region are labeled in blue. A magnified view of the active site of LTC4S within the dimer interface is shown to the right in which the DDM molecule has been removed for clarity. The predicted phosphorylation sites (Ser 36 , Ser 100 , Thr 40 , and Thr 41 ) are shown in that region together with the catalytic Arg 104 and a bound GSH (blue) molecule. in vitro and analyzed by autoradiography. A recombinant p70S6k was used for the in vitro phosphorylation assay as described (18). WT LTC4S was incubated with p70S6k in the presence of radioactive [␥-32 P]ATP in a reaction mixture as described under "Experimental Procedures." The phosphorylation of LTC4S was found to be detergent-specific. Enzyme purified with Triton X-100 as a detergent displayed a radioactive band with an M r of about 18,000, corresponding to phosphorylated LTC4S, whereas protein purified with the detergent n-dodecyl ␤-D-maltopyranoside (DDM) was not phosphorylated as judged by the faint radioactive band in the autoradiography (Fig. 3). In the crystal structure of LTC4S, a DDM molecule is bound in an intermonomeric, hydrophobic crevice believed to accommodate the substrate LTA 4 (Protein Data Bank code 2UUH; Fig. 1C). Thus, a possible explanation for the prevention of phosphorylation by DDM could be that the detergent molecule blocks the access of the kinases to the phosphorylation site(s). Based on the identification of Ser 36 as a phosphorylation site in LTC4S, we generated the mutant S36A and incubated it with p70S6k in the presence of radioactive [␥-32 P]ATP as with WT LTC4S. However, the results from autoradiography were not consistent and did not provide conclusive evidence to show that S36A mutant was not phosphorylated (data not shown). Thus, a radioactive band was consistently observed that could be due to the presence of an additional site(s), possibly Thr 40 , as indicated by MS analysis.
The Mutant S36E Displays Significantly Reduced LTC4S Activity-We next investigated the functional consequences of phosphorylation at Ser 36 by replacing this residue with a Glu (S36E), a common mimic of phosphoserine (25). The effects of mutation on LTC4S activity and kinetic parameters with its physiological substrates LTA 4 and GSH were assessed. Like-wise, the mutant T40E was constructed and functionally characterized. The specific activity of S36E LTC4S was reduced by nearly 80% (20 mol/min/mg) as compared with WT LTC4S (95 mol/min/mg), whereas for the T40E mutant (67 mol/ min/mg), the activity was reduced by 30% (Fig. 4A). The loss of activity of S36E agrees well with the previous observations of down-regulation of LTC4S activity by protein kinases (10,11). The steady-state kinetic parameters were determined for S36E and T40E mutants using LTA 4 as a substrate to test the effects on substrate binding and catalytic efficiencies (   could not be determined for S36E mutant as the formation of LTC 4 increased linearly with increasing concentrations of LTA 4 , indicating that substrate binding is very weak in this mutant. The apparent second order rate constant, k cat /K m LTA4 , which is the effective rate of substrate binding converted to product, was determined as 1.3 ϫ 10 5 M Ϫ1 s Ϫ1 , which is less than 10% of the efficiency displayed by WT LTC4S. In contrast, the catalytic properties of T40E were not changed significantly compared with WT enzyme. Thus, the catalytic turnover at saturating substrate concentration, k cat LTA4 , observed for WT and T40E LTC4S was 105 and 62 s Ϫ1 , respectively, whereas K m LTA4 remained in the same range for WT (76 M) and T40E (85 M). Therefore, WT and T40E enzyme displayed catalytic efficiencies, i.e. k cat /K m LTA4 , of 13.9 ϫ 10 5 and 7.7 ϫ 10 5 M Ϫ1 s Ϫ1 , respectively. Together, these data suggest that the active site architecture and enzyme-LTA 4 interactions have been affected due to the introduction of a negative charge at position 36.
The kinetic parameters for the peptide substrate GSH were similar to those of LTA 4 ( Table 1). The turnover rate by S36E (k cat GSH ) was reduced by 85% (3 Ϯ 0.1 s Ϫ1 ) compared with WT enzyme (19.0 Ϯ 1.0 s Ϫ1 ). The latter value is lower than k cat LTA4 due to the lower solubility and stability of LTA 4 compared with that of GSH. The values of K m GSH for S36E and T40E were a bit higher (120 and 220 M, respectively) compared with WT enzyme (70 M) ( Table 1). For T40E, the 3-fold increase in K m GSH may be explained by the results of MD simulations (see below).
Comparative Molecular Dynamics Suggests That a Phosphorylated Ser 36 Interacts with the Catalytic Arg 104 -We performed 100-ns molecular dynamics simulations, with snapshots taken every nanosecond, of LTC4S embedded in a lipid bilayer with and without phosphorylation at Ser 36 (Ser(P) 36 ). Analysis of the simulation snapshots indicated that the simulated systems generally appeared stable with energies and root mean square deviation values stabilizing after ϳ5 ns of simulation time (supplemental Fig. S2). The largest movements observed were in terminal regions distant from the site of phosphorylation and active site residues and were thus of limited functional relevance.
To identify functionally relevant changes in the motions of LTC4S upon phosphorylation, the dynamic cross-correlation matrix for each simulation was derived (26). To highlight phosphorylation-related differences in correlated movements, the dynamic cross-correlation matrix of native LTC4S was subtracted from the dynamic cross-correlation matrix of phosphorylated LTC4S and plotted as a heat map (supplemental Fig. S3). Two regions stood out in this map: the intersection between the first luminal loop (Arg 34 -Pro 43 , including the phosphorylation site) and residues located around the second luminal loop (Ala 89 -Ala 110 , including the end of helix 3 and beginning of helix 4) of the neighboring subunit and the intersection between the first luminal loop and residues in the beginning of helix 2 (Pro 43 -Phe 60 , i.e. right after the first luminal loop) of the neighboring subunit.
The obvious explanation for the changes highlighted by the dynamic cross-correlation matrix analysis is that phosphorylation of Ser 36 will pull this region toward spatially neighboring residues that provide complementary binding partners. Not all three subunits of LTC4S behaved exactly the same in this aspect, and the cross-subunit contacts were different for the three interfaces. This could be a real difference or just an effect of the limited time span (100 ns) of the simulation, but it is possible that a longer time course would equilibrate all structures at a more uniform state.
A detailed analysis of all simulation snapshots was performed to identify hydrogen bond (H-bond) partners of Ser 36 /Ser(P) 36 throughout the simulation time course (supplemental Table  S2). The most striking interactions were the H-bonds formed between Ser(P) 36 and Arg 104 (Fig. 5) that were present in 15-88% (depending on subunit interface) of all snapshots, which should be compared with Ͻ0.4% in the unphosphorylated model ( Table 2). This implies a high probability for formation of H-bonds between these residues upon phosphorylation. Importantly, Arg 104 is the key catalytic residue responsible for formation of the GSH thiolate anion (24). In addition, phosphorylation led to an increased frequency of H-bonding with Arg 34 in the same subunit. However, because Ser 36 and Arg 34 are very close (both spatially and in the primary sequence), this interaction is not likely to have significant functional effects. In addition, Arg 34 is not known to be important for catalysis or substrate binding.
Molecular dynamic simulation was also performed with a phosphorylated Thr 40 (Thr(P) 40 ) of LTC4S, which revealed a markedly pronounced interaction between Thr(P) 40 and Arg 51 of the neighboring subunit (data not shown). Arg 51 is an anchoring residue for the carboxyl group of the GSH (27) and was found to be not essential for catalysis (24), and its interaction with Thr(P) 40 may explain the increased K m GSH of T40E LTC4S as compared with WT enzyme (Table 1).
Crystal Structure of S36E LTC4S-A crystal structure of the mutant S36E was determined at 3-Å resolution (Protein Data Bank code 5HV9; supplemental Table S3), which indicates that the mutant was correctly folded and that the loss of activity is not due to the misfolded protein. The overall structure was similar to native LTC4S (Protein Data Bank code 2UUI) with one monomer in the asymmetric unit. Nine residues (Met Ϫ5 to Glu 4 ) and three residues (Pro 148 to Ala 150 ) were removed in the present structure at the N and C termini, respectively, because of the lack of density. Strong positive density was observed for the thiol group of GSH in the F o Ϫ F c difference map at 3 after initial refinement. Contouring F o Ϫ F c map to 2.5 resulted in a positive curved density representing GSH at the active site. The GSH molecule was fitted, and complete density was achieved at 1 of 2F o Ϫ F c map. A minor positional shift for the thiol group of GSH accompanied by an increased distance (0.7 Å) to Arg 104 was observed at the active site (Fig. 6) compared with the structure of WT LTC4S with bound GSH (Protein Data Bank code 2UUH), which corroborates the results of MD simulations and provides a structural basis for the reduced catalytic activity displayed by S36E. Moreover, no positive density for a DDM molecule (mimics LTA 4 in 2UUH) was detected in the putative active site of the S36E structure, suggesting that lipid substrate binding is compromised.
Phosphomimetic Mutant S36E Is Less Sensitive to a Synthetic Inhibitor-A recent study suggests that protein phosphorylation may affect drug inhibitor binding to target proteins (28). They classified two types of mechanisms by which phosphorylation affects drug efficacy. In one type, phosphorylation inhibits both drug binding and target activity, whereas in the other type, phosphorylation inhibits drug binding while increasing target activity (28). Here, we used the phosphomimetic mutant S36E to test the effect of phosphorylation on inhibitor binding to LTC4S. In a previous study, a nanomolar inhibitor, TK04, was used to probe the inhibition of human and mouse LTC4S (29). The inhibitor (0.05-15 M) was incubated with the same amount of WT and S36E enzyme (0.1 g) at a fixed concentration of LTA 4 (20 M) and GSH (5 mM). TK04 was less efficient in inhibiting the S36E mutant (IC 50 ϭ 389 Ϯ 69 nM) at low inhibitor concentrations (ՅIC 50 ) compared with WT enzyme (IC 50 ϭ 211 Ϯ 52 nM), whereas at higher concentrations there were no significant differences (Fig. 7). The observed behavior with the S36E mutant indicates that phosphorylation at Ser 36 may interfere with TK04 binding, thus effecting the inhibitor potency. TK04 was proposed to occupy the LTA 4 binding site and interact with Arg 104 based on molecular docking results (30). The efficiency of the TK04 inhibitor at concentrations around or below IC 50 seems reduced with the phosphomimetic S36E mutant, which is yet an indication that phosphorylation affects lipid substrate binding.
Concluding Remarks-We have used MS/MS analysis and site-directed mutagenesis to identify Ser 36 as a predominant and functionally important p70S6k phosphorylation site in LTC4S. An alternative site, Thr 40 , was also identified at low frequency and was found to be of marginal functional relevance. Results of comparative MD simulations and x-ray crystallography indicate that phosphorylation of Ser 36 acts by disturbing the catalytic action of Arg 104 and reducing substrate access to the active site (Fig. 8). P70S6k-dependent LTC4S phosphorylation was demonstrated in monocytes (18). Given the complexity of PKC-mediated protein phosphory-

Phosphorylation of Leukotriene C 4 Synthase
lation events, residues other than Ser 36 may be functional phosphorylation sites involving other kinases in other cellular contexts. Such possibilities deserve further studies and may give additional insights into the mechanisms of LTC4S phosphoregulation.

Experimental Procedures
Chemicals, Reagents, and Enzymes-GSH, 2-mercaptoethanol, imidazole, Tris base, NaCl, Triton X-100, and sodium deoxycholate were obtained from Sigma. DDM was purchased from Anatrace. [␥-32 P]ATP was ordered from PerkinElmer Life Sciences. p70S6k, Mg-ATP mixture, and kinase assay dilution buffer were purchased from Merck-Millipore. Pepsin, trypsin, and chymotrypsin were obtained from Promega (Madison, WI). Protease and phosphatase inhibitor mixtures were from Sigma and Thermo Scientific, respectively. LTA 4 was purchased from Cayman as LTA 4 methyl ester and further converted to LTA 4 by saponification as described previously (31).
Site-directed Mutagenesis-Site-directed mutagenesis was performed according to the QuikChange protocol (Stratagene, La Jolla, CA). WT LTC4S cDNA with an additional N-terminal His 6 tag was subcloned into pPICZA (Invitrogen) vector and used as a template to generate all other mutants using the primers listed in supplemental Table S1. To check the mutations and other nonspecific changes, the protein-coding part of the plas-mid vectors was verified by DNA sequencing from SEQLAB, Göttingen, Germany.
Protein Expression and Purification-WT LTC4S and all mutants were expressed in yeast Pichia pastoris, and the purification was performed in a single step on an S-hexylglutathione-agarose column (24). The protein used for the in vitro phosphorylation assay was desalted on a PD-10 column to exchange buffer with 20 mM HEPES (pH 7.4) containing 0.1 mM dithiothreitol (DTT) and 0.05% Triton X-100. Conversely, the protein prepared for crystallization was further purified on a Superdex 200 16/60 (GE Healthcare) with 20 mM Tris (pH 8.0), 100 mM NaCl, 0.03% DDM (w/v), and 0.5 mM tris(2-carboxyethyl)phosphine. Protein concentration was determined by the Lowry method (32) or the Pierce TM BCA Protein Assay kit followed by SDS-PAGE on a Phast system (GE Healthcare).
Identification of Phosphorylation Site(s) by Mass Spectrometry-In vitro phosphorylated WT LTC4S was separated using one-dimensional PAGE and stained with Coomassie Brilliant Blue. The protein band was excised manually and digested in gel using a MassPREP robotic protein-handling system (Waters, Millford, MA) according to the manufacturer's instructions. After reduction with DTT and alkylation with iodoacetamide, the proteins were digested with 0.3 g of trypsin (modified; Promega) in 50 mM ammonium bicarbonate for 5 h at 40°C. The tryptic peptides were extracted with 1% formic acid and 2% acetonitrile followed by 50% acetonitrile twice. Phosphopeptides were further enriched on a PhosphoCatch TM microspin column (Promega) loaded with a combination of zirconium and titanium oxide resins.
The in vitro phosphorylated samples were digested in solution as described previously (33). The proteins were digested overnight in 50 mM ammonium bicarbonate, 30% DMSO, and trypsin (at a ratio of 1:20 trypsin:protein; modified) at 37°C.
Peptides from both in-gel and in-solution digestion were desalted using ZipTips (C 18 ; Merck Millipore Ltd., Ireland) followed by separation using online nano-scale-LC-MS/MS (reversed phase C 18 ) and analyzed on an LTQ Velos Orbitrap electron transfer dissociation mass spectrometer (Thermo Fisher Scientific, Germany). A 40-min gradient of buffer A and B (A, 0.1% formic acid in water; B, 0.1% formic acid in acetonitrile) was used for the separation as follows: 5-30% B in 35 min followed by 30 -95% B in 5 min. The flow rate was 300 nl/min. MS spectra were acquired at a resolution of 60,000 followed by fragmentation of the five most intense peaks. The peptides were  either fragmented by only collision-induced dissociation or by higher energy C-trap dissociation first followed by electron transfer dissociation of the same precursor.
Mass lists extracted by Raw2MGF v2.1.3 (34) were searched against the Swiss-Prot database (downloaded February 7, 2014) using Mascot search engine v2.3.02 (Matrix Science Ltd., London, UK). The WT and mutant constructs used in this study were also added to the database. The following parameters were used for the database searching: tryptic digestion (with a maximum of two miscleavages); carbamidomethylation (Cys) as fixed modification; oxidation (Met), pyroglutamate (Gln), deamidation (Asn/Gln), and phosphorylation (Ser/Thr/Tyr) as variable modifications; 10 ppm as precursor tolerance; and 0.25 Da as fragment tolerance.
Enzyme Activity Assay-Formation of the enzyme product LTC 4 was measured by UV absorbance at 280 nm using high performance liquid chromatography (HPLC) as described earlier (24). Enzyme (0.1 g) together with GSH (5 mM) was incubated in the presence of LTA 4 (30 M) for 15 s at room temperature in a 100-l reaction volume and terminated by adding 200 l of methanol to the reaction mixture followed by the addition of prostaglandin B 2 as an internal standard. The reaction buffer contained 25 mM Tris-HCl (pH 7.8), 0.05% Triton X-100, and 5 mM 2-mercaptoethanol. The steady-state kinetic parameters were determined by varying the LTA 4 concentration from 10 to 120 M while keeping the GSH concentration at 5 mM. Alternatively, the GSH concentration was varied between 0.05 and 4 mM while the concentration of LTA 4 was kept at 30 M. The kinetic data were fitted to the Michaelis-Menten equation using non-linear regression in GraphPad Prism to extract all the kinetic parameters. The k cat /K m was determined using RFFIT in SIMFIT.
Enzyme Inhibition Assay-Inhibition studies of WT and S36E LTC4S with the inhibitor TK04 were performed using an assay in a 96-well format as described earlier (29) to determine the inhibition parameters. The TK04 concentration was varied between 0 and 15 M while keeping the LTA 4 concentration constant at 20 M in the presence of 5 mM GSH and 0.1 g of enzyme. The data were analyzed with non-linear regression using GraphPad Prism to calculate the IC 50 values.
Molecular Dynamics-For molecular dynamics, the software YASARA Structure was used (35). The crystal structures of human LTC4S with bound GSH (Protein Data Bank code 2UUH with His tag removed and stripped of all water and ligands other than GSH) and with modeled hydrogens in H-bond-optimized positions (36) were used in the simulations. The trimeric form of the enzyme was generated from the information in the Protein Data Bank file. Phosphorylation of Ser 36 and Thr 40 was manually built. The AMBER03 force field (37) was used, and force field parameters for non-standard protein residues were generated with YASARA'S built-in AutoSMILES algorithm (38,39).
By using the default protocol for simulation of membrane proteins in YASARA (YASARA macro for running a molecular dynamics simulation of a membrane protein with normal or fast speed), the protein was embedded in a phosphatidylethanolamine lipid bilayer and slowly adapted (including deletion of clashing protein-membrane residues and energy minimization) to accommodate the inserted protein. Subsequently, water molecules were added, pK a values were assigned, the whole simulation cell was neutralized by addition of sodium (at locations of lowest electrostatic potential) and chloride (at locations of highest electrostatic potential) ions to a final concentration of 154 mM, and the system was energy-minimized (40).  36 ]LTC4S was generated from the crystal structure of LTC4S (Protein Data Bank code 2UUH) where Ser 36 was exchanged for phosphoserine in Coot followed by geometry minimization using the PHENIX geometry minimization tool. Finally, the figure was prepared using PyMOL.