Human Leukotriene C4 Synthase at 4.5 A˚Resolution in Projection

Human Leukotriene C4 Synthase at 4.5 A˚ Resolution in Projection

Ingeborg Schmidt-Krey,1,* Yoshihide Kanaoka,2 MGST3, FLAP, and microsomal prostaglandin E syn- Deryck J. Mills,1 Daisuke Irikura,2 thase-1 (Jakobsson et al., 1999). The amino acid se- 1 2 2 Winfried Haase, Bing K. Lam, K. Frank Austen, quence of the human LTC4S is 44%, 31%, and 27% and Werner Ku¨ hlbrandt1 identical to that of human MGST2, FLAP, and MGST3, 1Department of Structural Biology respectively, and has less similarity to that of MGST1 Max-Planck-Institute of Biophysics (18% identity) and microsomal prostaglandin E syn- Marie-Curie-Strasse 15 thase-1 (14% identity).

60439 Frankfurt am Main LTC4S shows overlapping and distinct enzymatic Germany properties as compared to other members of the MAPEG 2 Department of Medicine family. MGST2 (Jakobsson et al., 1996) and MGST3 (Ja-

Harvard Medical School and kobsson et al., 1997) can also conjugate LTA4 as well Division of Rheumatology, Immunology, and Allergy as xenobiotics with reduced glutathione in vitro, while

Brigham and Women’s Hospital LTC4S has strict substrate specificity for LTA4 (Nicholson 2ϩ One Jimmy Fund Way et al., 1993). LTC4S activity is augmented by Mg ions Boston, Massachusetts 02115 and phosphatidylcholine and is inhibited by Co2ϩ ions (Nicholson et al., 1992a). N-ethylmaleimide, a sulfhydryl group reactive agent, activates MGST1 (Morgenstern et Summary al., 1980) but inhibits LTC4S activity in the human leuke- mic monoblast cell line, U937 (Nicholson et al., 1992b)

and the recombinant human LTC4S (B.K.L., unpublished Leukotriene (LT) C4 synthase, an 18 kDa integral mem- results). LTC4S is inhibited by a FLAP inhibitor, MK-886, brane enzyme, conjugates LTA4 with reduced glutathi- ␮M (Lam et al., 1996). Site-directed 3ف with an IC50 of one to form LTC4, the parent compound of all cysteinyl leukotrienes that play a crucial role in the pathobiology mutagenesis of human LTC4S suggests that amino acids of bronchial asthma. We have calculated a projection V49, A52, N55, Y59, Y93, Y97 and I27, V35, V49, A112 are involved in glutathione and LTA4 binding, respec- map of recombinant human LTC4 synthase at a resolution of 4.5 A˚ by electron crystallography, which shows tively, and that R51 and Y93 are crucial for opening the that the enzyme is a trimer. A map truncated at 7.5 A˚ epoxide ring of LTA4 and for activating the thiolate anion visualizes four transmembrane ␣ helices per protein of glutathione, respectively (Lam et al., 1997; B.K.L., monomer. The densities in projection indicate that unpublished data). most of the ␣ helices run nearly perpendicular to the Gel filtration chromatography suggests that LTC4S is enzymatically active as a homodimer (Nicholson et plane of the membrane. At this resolution, LTC4 synthase is strikingly similar to microsomal glutathione al., 1993). Bioluminescence resonance energy transfer S-transferase 1, which belongs to the same gene fam- studies in living cells demonstrated that LTC4S forms a ily but bears little sequence identity and no resem- homooligomer (Svartz et al., 2003), and fluorescence resonance energy transfer and crosslinking studies sug- blance in substrate specificity to the LTC4 synthase. These results provide new insight into the structure gest that LTC4S can form a hetero-dimer or a hetero- and function of membrane proteins involved in eicosa- trimer with FLAP (Mandal et al., 2004). noid and glutathione metabolism. To elucidate the structure of LTC4S, we have overexpressed human LTC4S in fission yeast, purified the enzyme by S-hexylglutathione affinity chromatography, Introduction and grown two-dimensional (2D) crystals. Electron crystallography of 2D crystals has evolved into a viable Leukotriene (LT) C synthase (LTC S) is an 18 kDa inte- 4 4 method to solve structures of membrane and soluble gral perinuclear membrane enzyme that conjugates proteins at atomic or near atomic resolution (Henderson LTA , an unstable epoxide formed from arachidonic acid 4 et al., 1990; Ku¨ hlbrandt et al., 1994; Nogales et al., 1998; by 5-lipoxygenase in the presence of 5-lipoxygenase- Murata et al., 2000; Ren et al., 2001; Miyazawa et al., activating protein (FLAP), with reduced glutathione to 2003; Gonen et al., 2004). LTC4S can be induced to form LTC4 (Nicholson et al., 1993; Yoshimoto et al., form well-ordered 2D crystals when the purified and 1988). LTC4 and its metabolites, LTD4 and LTE4, are col- delipidated enzyme is reconstituted into phospholipid lectively called cysteinyl leukotrienes and are implicated bilayers. 2D crystals yielded amplitudes and phases of in the pathobiology of allergic and inflammatory dis- structure factors to approximately 4.5 A˚ , of which a eases, including bronchial asthma and fibrosis (Lewis projection map was calculated. A comparison of the et al., 1980; Murphy et al., 1979). The cloning of the projection structure of LTC4S to a distantly related mem- human LTC4S cDNA (Lam et al., 1994; Welsch et al., ber of the same protein superfamily revealed striking 1994) revealed that LTC4S belongs to the MAPEG (mem- similarities and marked differences. brane-associated proteins with functions in eicosanoid and glutathione metabolism) superfamily, including mi- Results and Discussion crosomal glutathione S-transferase 1 (MGST1), MGST2,

Human LTC4S was overexpressed in fission yeast and *Correspondence: [email protected] purified to apparent homogeneity. The yield of the re- Structure 2010

Figure 1. Crystal Morphology and Projection Data of LTC4S

(A) A negatively stained crystalline leukotriene C4 synthase membrane sheet. The lattice is very difficult to distinguish even on images from negatively stained crystals at high magnification, since the protein is mostly hydrophobic. The scale bar is 1 ␮m. (B) The combined phase error of individual reflections of the merged data set including four images. The sizes of the boxes represent the phase error with 1 Ͻ 8Њ,2Ͻ 14Њ,3Ͻ 20Њ,4Ͻ 30Њ,5Ͻ 40Њ,6Ͻ 50Њ,7Ͻ 70Њ, and 8 Ͻ 90Њ. The larger boxes are labeled. Only the unique section of reflections within reciprocal space in plane group symmetry p321 is displayed.

(C) The projection map of human leukotriene C4 synthase at 4.5 A˚ resolution showing one unit cell. The crystals have p321 symmetry, and the 2-fold axes in projection are indicated by arrows. Thus, the unit cell contains two trimers (indicated by dashed and dotted lines) in the two possible membrane orientations. The unit cell dimensions are a ϭ b ϭ 73.4 A˚ , ␥ϭ120Њ.

combinant protein was approximately 2.5 mg per liter scan images of LTC4S crystals embedded in trehalose culture. Enzymatic properties of the recombinant LTC4S (Table 1). Image amplitudes and phases extend to a were essentially identical to those of the native enzyme resolution of approximately 4.5 A˚ (Table 2; Figure 1B); purified from human lung (Penrose et al., 1992) and the the highest resolution of a recombinant eukaryotic mem- recombinant enzyme from transfected COS cells (Lam brane protein obtained to date. The enzyme forms crys- et al., 1997). Following reconstitution with minimal tals with a lattice of plane group symmetry p321 (Table amounts of dimyristoyl phosphatidyl choline (DMPC), 3) with unit cell dimensions of a ϭ b ϭ 73.4 A˚ , ␥ϭ120Њ the recombinant human LTC4S reproducibly forms 2D (Figure 1C). The unit cell contains two closely packed crystals in membrane sheets and vesicles measuring up protein trimers with opposite membrane orientations. to several microns in diameter (Figure 1A). A projection While LTC4S was predicted to form dimers (Nicholson map was calculated using merged data from four spot- et al., 1993), the projection map clearly reveals a trimer. Projection of Human Leukotriene C4 Synthase 2011

˚ Table 1. Electron Crystallographic Data spacing of approximately 10 A between adjacent densities, which is indicative of transmembrane ␣ helices Preparation running roughly perpendicular to the plane of the mem- Stabilizing medium trehalose brane. Consequently, the protein monomer is consti- ␣ Imaging Conditions tuted of four transmembrane helices. Each monomer contributes two ␣ helices (labeled C and B in Figure 2E) Nominal magnification 70,000 to a triangular arrangement of six ␣ helices in the center High tension (kV) 300 of the trimer (outlined by a dashed red circle in Figure Electron source field emission Dose (eϪ/A˚ 2)20 2E). Two of these inner (C and B) and two of the outer Temperature (K) 4 ␣ helices (A and D) each form the monomer. At this stage, Defocus range (A˚ ) 4,600–11,800 it is not known which pair of inner ␣ helices belongs to Image Processing the monomer: either C and B1 or C and B2. One of the outer ␣ helices, D, has a center-to-center Scan area (pixels) 4,000 ϫ 4,000 ˚ ␮ spacing of approximately 10 A with the corresponding Pixel size ( m) 7 ␣ Program system MRC programs helix in the second mirrored trimer within the unit No. images merged 4 cell. The close spacing suggests protein-protein crystal Temperature factors contacts of ␣ helices in opposite membrane orientations 4.5 A˚ map: (A˚ 2) Ϫ150 rather than protein-lipid-protein contacts to be respon- 7.5 A˚ map: (A˚ 2) Ϫ200 sible for the crystal formation. This explains the low Crystal Parameters amount of phospholipid required for 2D crystal formation. Unit cell a ϭ b ϭ 73.4 A˚ , ␥ϭ120Њ Projection symmetry p321 Even though the trimer-trimer crystal contacts between the two adjacent monomers in the center of the unit cell might be thought of as constituting a dimer, these monomers have opposite membrane orientations The dimensions of one monomer are approximately 20 due to the p321 symmetry of the crystals. Thus, it would by 22 A˚ , and the trimer has a diameter of about 43 A˚ . not be feasible from a biological point of view to interpret To visualize possible ␣ helices, the data were trun- the map as showing protein dimers. Furthermore, a strik- cated at a resolution of 7.5 A˚ (Figure 2). In a projection ing similarity of the overall arrangement of the secondary map, this resolution usually allows the identification of and quarternary structure with MGST1 provides addi- ␣ helices that run perpendicular to or are only slightly tional support for a trimeric nature of LTC S. inclined in respect to the plane of the membrane. At a 4 LTC4S belongs to the MAPEG protein superfamily (Ja- higher resolution, a projection map is often more difficult kobsson et al., 1999), as does rat liver MGST1, of which to interpret due to the large amount of superposed detail a preliminary three-dimensional (3D) structure has been present (as discussed below). Each trimer is constituted calculated to a resolution of 6 A˚ (Schmidt-Krey et al., of 12 nearly circular densities with a center-to-center 2000; Holm et al., 2002). While both enzymes are capable of conjugating reduced glutathione with their sub-

strates, LTC4S possesses strict substrate specificity for Table 2. Phase Residuals for Data Merged from Four Images leukotriene A . In contrast, MGST1 completely lacks this (Up to IQ Better or Equal to 7) 4 substrate specificity and is known for conjugating a par- Resolution Range (A˚ ) No. of Reflections Phase Residual (⌬φЊ) ticularly wide range of compounds with glutathione in 100–10.1 75 6.1 liver detoxication. Furthermore, the kinetics of the two –7.1 65 11.4 enzymes differ significantly, and they only share minimal –5.8 50 26.4 sequence identity (Figure 3A; Jakobsson et al., 1999). –5.0 45 33.0 A comparison of hydrophobicity plots has indicated a –4.5 49 31.8 similar topology of the members of the MAPEG super- 100–4.5 284 20.9 family (Jakobsson et al., 1999). The 3D structure of

Table 3. Phase Residuals in Different Two-Sided Plane Groups for One Image Phase Residual versus Target Residual Based on Two-Sided Other Spots No. of Phase Residual versus No. of Statistics Taking Friedel Plane Group (90Њ Random) Comparisons Theoretical (45Њ Random) Spots Weight into Account p1 16.7 172 12.0 172 24.0 p2 72.0 86 36.0 172 17.4 c12_b 67.3 40 67.7 8 17.3 c12_a 64.0 39 45.7 6 17.4 p3 18.8a 138 — — 16.7 p312 55.4 345 67.5 26 17.0 p321 19.4a 344 15.8 24 17.0 p6 55.6 362 40.2 172 18.4 p622 52.6 775 40.2 172 17.5 aAcceptable suggestions based on the statistics. Structure 2012

Figure 2. Comparison of the LTC4S and MGST1 Projection Structures (A–D) The projection maps of the (A) leuko-

triene C4 synthase (LTC4S) and the (B) microsomal glutathione S-transferase 1 (MGST1) trimers at 4.5 A˚ and maps of the same data

sets of (C) LTC4S and (D) MGST1 truncated at 7.5 A˚ . The maps were truncated at 7.5 A˚ resolution to visualize densities corresponding to ␣ helices more clearly. The scale bar is 10 A˚ . (E) The projection map at 7.5 A˚ of MGST1 (red

contour lines) superimposed on the LTC4S trimer. One trimer is marked with a red dashed circle. Unique densities corresponding to ␣ helices are labeled A–D (see main text for explanation). While the overall arrangement of the ␣ helices is very similar, and identical in the case of ␣-helix C, differences can be observed in the position and size of densities A, B, and D. At this point, it is not possible to outline the monomer, and either ␣ helix B1 or helix B2 could constitute the monomer in combination with ␣ helices A, C, and D. The unit cell dimensions are a ϭ b ϭ 73.4 A˚ , ϭ120Њ.

MGST1 revealed four ␣ helices per protein monomer At a resolution of 4.5 A˚ (Figures 2A and 2B), the ␣ (Schmidt-Krey et al., 2000). One of these ␣ helices could helices of the trimers cannot be recognized, and it only not be identified in projection at resolutions better than becomes possible to identify the outer, more highly tilted 4A˚ (Hebert et al., 1995; Schmidt-Krey et al., 1999) due ␣ helices at approximately 7.5 A˚ (Figures 2C and 2D). to its relatively high tilt of 27Њ with respect to the plane When interpreting projection maps at high and interme- of the membrane (Schmidt-Krey et al., 2000). By calcu- diate resolution, it thus appears generally advisable to lating projection maps at an intermediate resolution of calculate projection maps of highly ordered 2D crystals 7.5 A˚ , we could resolve the fourth helix in the rat liver not only to the high-resolution limit, but also to a resolu- MGST1 monomer and compare the projection structure tion cut-off in the intermediate range of 7.5–10 A˚ .Asin with the human LTC4S monomer. To determine at which the case of MGST1 and LTC4S, a projection map at lower resolution the more highly tilted ␣ helix can be identified resolution might provide more information in projection in projection, maps at resolution cut-offs in the range because the secondary structure is not obscured by of 3.5 A˚ to 15 A˚ of a projection data set extending to higher resolution detail.

3A˚ were calculated (data not shown). The MGST1 rather LTC4S and MGST1 show a striking similarity in the than the LTC4S data were used, since we could verify overall arrangement of their secondary and quarternary our conclusions from the projection with the known 3D structure (Figure 2E). The inner ␣ helices of the LTC4S map. The projection maps of LTC4S and MGST1 are not trimer are positioned almost exactly like the correspond- comparable at higher resolution, since the proteins only ing ␣ helices in the MGST1 trimer in a triangular arrange- possess very limited sequence identity. ment. Yet the inner density B in the LTC4S trimer has Projection of Human Leukotriene C4 Synthase 2013

differences in inclination and placement of ␣ helices in the two enzymes.

Experimental Procedures

Overexpression, Purification, and Crystallization

An NdeI restriction site and His6-tag were created by PCR at the

initiating ATG codon and the C terminus of the human LTC4S cDNA (Lam et al., 1994), respectively, by using a sense primer (5Ј-GGTCAT ATGAAGGACGAGGTAGCT-3Ј) and an antisense primer (5Ј-CTTGAA TTCAGTGATGGTGATGGTGATGGGCCCACGGCAGCAGCGT-3Ј). The amplified fragment was subcloned into a pCR-Script vector (Stratagene, La Jolla, CA), and the sequence was confirmed by DNA sequencing. The NdeI-SmaI fragment from the verified plasmid was then subcloned into a pESP-3 vector (Stratagene) that had been digested with NdeI and SmaI to remove the glutathione S-transferase gene derived from the pESP-3. The resultant plasmid was transfected into Shizosaccharomyces pombe, h- leu1-32, and stable

clones were established. Recombinant human LTC4S protein was induced by depletion of thiamine in the culture media according to the manufacturer’s instructions and purified according to Penrose et al. (1992) with some modification. Briefly, the yeast pellets were disrupted with glass beads in 50 mM HEPES buffer (pH 7.9) with 1 mM EDTA, 10 mM 2-mercaptoethanol, and 10% glycerol. The homogenates were solubilized by adding Triton X-100 (final 1%) and sodium deoxycholate (0.5%) and bound to S-hexylglutathione agarose (Sigma, St. Louis, MO). The agarose was washed with 50 mM HEPES (pH 7.6) containing 1 mM 2-mercaptoethanol, 10% glycerol, 1% TritonX-100, and 0.5% sodium deoxycholate, and the

LTC4S was eluted with 50 mM HEPES (pH 7.6) containing 1 mM glutathione, 10% glycerol, 0.1% Triton X-100, 0.5% sodium deoxycholate, and 30 mM probenecid (Sigma). The eluates were then concentrated about 10-fold by a 10 kDa cut-off centrifugal filtration Figure 3. Membrane Topology and Active Site (Millipore, Billerica, MA). The purity and quantity of the enzyme were assessed with SDS-PAGE and subsequently stained with Coomas- (A) An amino acid sequence alignment of LTC S and MGST1. Identi- 4 sie brilliant blue with bovine serum albumin as a standard. The cal residues are boxed in gray, and residues important in the active enzymatic properties of the recombinant protein were measured site are boxed in black. according to Lam et al. (1997). The purified and delipidated protein (B) Schematic representation of the membrane topology of LTC S 4 (as monitored by thin layer chromatography) at a concentration and MGST1. Residues significant in the active site of LTC S are 4 of 1mg/ml was crystallized by dialyzing a protein-detergent-lipid labeled in black, while the analogous residues in MGST1 are labeled mixture containing dimyristoyl phosphatidyl choline (DMPC) solubi- in white. lized in 1% Triton X-100 at a molar lipid-to-protein-ratio of 1 against detergent-free buffer (50 mM HEPES [pH 7.6] containing 1 mM EDTA, 10 mM 2-mercaptoethanol, 20% glycerol, 10 mM GSH, and an oval shape, which might be due to a higher tilt of the 50 mM KCl) for 10–14 days at room temperature. ␣ helix in respect to the membrane. On the other hand, Electron Microscopy and Image Processing ␣ the outer helices of LTC4S appear less elongated when Specimens negatively stained with 1% uranyl acetate were screened comparing them to ␣ helices A and D of MGST1 for crystals using Phillips CM12 and CM120 electron microscopes (Schmidt-Krey et al., 2000). This might be an indication equipped with 2Kx2K Gatan CCD cameras. Samples that contained of outer ␣ helices that are not as highly tilted as in large and crystalline membranes were mixed with 4% trehalose as MGST1. the embedding medium for the back-injection method (Wang and Ku¨ hlbrandt, 1991). The crystals were incubated for 60 s on copper Another member of the MAPEG superfamily, micro- or molybdenum grids, blotted on two layers of Whatmann #4 filter somal prostaglandin E synthase-1, also shows a trimer paper, and plunged into liquid nitrogen. Spot-scan images on Kodak in a projection structure at 10 A˚ (Thore´ n et al., 2003). SO-163 film (45 ms/spot exposure time) were collected with a JEOL Details beyond the trimeric nature of the microsomal 3000SFF electron microscope at an accelerating voltage of 300 kV, prostaglandin E synthase-1 are not visible and thus can- a specimen temperature of 4 K (Fujiyoshi et al., 1991; Fujiyoshi, 1998), and a nominal magnification of 70,000. The best images were not be compared. selected by optical diffraction. Areas of 4000 ϫ 4000 pixels were In LTC4S, a Y93 was identified as the residue responsi- scanned with a Zeiss SCAI scanner at a step size of 7 ␮m. The ble for the activation of the thiolate anion of glutathione images were processed with the MRC programs according to Hen- (Lam et al., 1997) and is conserved in the MAPEG super- derson et al. (1986) and Crowther et al. (1996). For the comparison family (Jakobsson et al., 1999). In addition, LTC S con- of MGST1 projection maps, the same data set (containing only image 4 data) was used and truncated at various resolutions under otherwise tains an R51 that is thought to be the proton donor for identical conditions. The orientation of the MGST1 trimer for super- the opening of the LTA4 epoxide ring. The corresponding position on the map of LTC4S was determined by comparing super- residue in MGST1 is C49. Both the R51 and the C49 can positions of both membrane orientations (data not shown). be located to the first loop, and with the Y93 in the third The sequence alignment was calculated with CLUSTAL W (1.82). loop, both loops would be placed on the same side Acknowledgments of the membrane (Figure 3B). A 3D model at higher resolution will reveal more about these loops and the We thank Dr. Janet Vonck for valuable discussions and Vijay Patel active site as well as provide an explanation for the for help in preparing this manuscript. I.S.-K. gratefully acknowledges Structure 2014

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