18 C Cr ry ys st ta al l S St tr ru uc ct tu ur re e o of f H Hu um ma an n M Me em mb br ra an ne e- -i in nt te eg gr ra at te ed d P Pr ro ot te ei in n R Re es sp po on ns si ib bl le e f fo or r B Bi io os sy yn nt th he es si is s o of f C Cy ys st te ei in ny yl l L Le eu uk ko ot tr ri ie en ne es s The human membrane-integrated protein leukotriene C 4 synthase (LTC 4 S) embedded in the nuclear membrane is the first enzyme involved in the biosynthesis of cysteinyl leukotriene (Cys-LT), and its inhibitor is expected to be a new therapeutic agent for symptoms caused by proinflammatory acute smooth muscle constriction in conditions such as anaphylaxis, allergy, and asthma. LTC 4 S conjugates leukotriene (LT) A 4 and reduced glutathione (GSH) to synthesize LTC 4 (Fig. 1). LTC 4 and its metabolites, LTD 4 and LTE 4 , are collectively called Cys-LTs, and Cys-LTs have been known as slow-reacting substance of anaphylaxis (SRS-A) [1,2]. The smooth muscle constriction activity of Cys-LT is one thousand-fold higher than that of histamine, a well-known inflammatory substance. LTC 4 S is a potential target in the development of new therapeutic agents for anaphylaxis, allergy, and asthma, and the three- dimensional structure of LTC 4 S enables us to apply modern method for drug discovery. We elucidated the crystal structure of human LTC 4 S at 3.3 Å resolution using beamline BL44B2 , which makes human LTC 4 S one of three human membrane-integrated proteins whose structures were determined crystallographically first [3]. Indeed, one of the bottlenecks is the production of a fully active human membrane-integrated protein at crystallographic quality and quantity using a heterologous expression system. We established a heterologous expression system using the fission yeast Schizosaccharomyces pombe to obtain a fully active LTC 4 S on the milligram scale from a few liters of yeast culture. The crystal structure of LTC 4 S revealed that trimeric LTC 4 S in a three-fold symmetry is a biological functional unit [3]. An LTC 4 S monomer is composed of five α -helices, the first four of which are transmembrane α -helices and the fifth α -helix protruding into the bulk solvent from nuclear membrane (Fig. 2(a,b)). The V-shaped cleft surrounded by five α -helices is the substrate binding site. All the five α -helices contribute to the binding of a unique U-shaped GSH to the upper side of the cleft (Fig. 2(c)). For example, the positive charge of Arg51 of α -helix II is essential to the binding of GSH [4]. The remaining space extending from GSH to the bottom of the cleft would be the binding site of LTA 4 . The depth and shape of the space fits well to the molecular shape of the aliphatic chain of LTA 4 having two cis- double bonds at C11 and C14 (Fig. 3). Indeed, the hexyl moiety of S-hexyl glutathione as a weak inhibitor occupies the space in the S-hexyl GSH complex crystal (Fig. 2(d)). Furthermore, the aliphatic chain of dodecyl maltoside occupies the space in the GSH complex crystal. Together with these results, the active site is located between the two adjacent monomers in the trimeric LTC 4 S, and the trimeric LTC 4 S is the biological functional unit. The crystal structure of LTC 4 S shows that the conjugation of LTA 4 and GSH is induced by acid-base catalysis, in which Arg31 and Arg104 are the acid and base, respectively. During the course of the conjugation, LTC 4 S activates the thiol group of bound GSH for the nucleophilic attack of the sixth carbon of LTA 4 and provide a hydrogen to the negatively charged oxygen derived from the epoxide group at the fifth carbon. The side chain of Arg104 as the base decreases the pKa of the thiol group of GSH as the reactive species under the physiological condition due to a hydrogen bond between Arg104 and the thiol group of GSH (Fig. 2(c)). When LTA 4 binds to the GSH-binding LTC 4 S (Fig. 3), the activated thiol group would attack C6 on the opposite side against the epoxy oxygen of LTA 4 , and a negative charge would grow on the epoxy oxygen concurrently with the breaking of the bond between the epoxy oxygen and C6. Finally, Arg31 as the acid provides a proton to the negatively charged oxygen at C5, resulting in LTC 4 as the conjugated product. The crystal structure of the human membrane- C11 C6 LTA 4 LTC 4 GSH O O O O Gly S OH OH H N Glu OH LTC 4S Fig. 1. Biosynthesis of Cys-LT. LTC 4 S catalyzes conjugation between LTA 4 and reduced GSH to synthesize LTC 4 , which is further metabolized to LTE 4 through LTD 4 by hydrolytic enzymes. Life science : Structural Biology 19 integrated protein LTC 4 S reveals unique features for the strict substrate specificity and the conjugation catalyzed by two arginine residues in an acid-base- catalytic manner. We hope that this work contributes to the advancement of the pathobiology of inflammatory diseases and to the development of new therapeutics. Hideo Ago* and Masashi Miyano SPring-8 / RIKEN *E-mail: ago@spring8.or.jp References [1] S.-E. Dahlén et al .: Nature 288 (1980) 484. [2] K.F. Austen: Prostaglandins Other Lipid Mediat. 83 (2007) 182. [3] H. Ago, Y. Kanaoka, D. Irikura, B.K. Lam, T. Shimamura, K.F. Austen and M. Miyano: Nature 448 (2007) 609 . [4] B.K. Lam et al .: J. Biol. Chem. 272 (1997) 13923. (a) (b) (c) (d) Fig. 2. Crystal structure of LTC 4 S. (a,b) Schematic representations of trimeric LTC 4 S. α -Helices from a monomer are tube models in rainbow color, and the bound GSH is in space-filling model. In all panels, the sequential number on each α -helix is the order of the α -helix from the N-terminus. The small letter following the sequential number means that the α -helix is subdivided. A small capital in square brackets corresponds to the monomer, which contains the α -helix. (c) Close-up view of the bound GSH. Dashed lines are polar interactions participating in GSH binding. (d) Electron density of inhibitor S -hexyl GSH at 5 Å resolution. Fig. 3. LTA 4 binding model. The stick model with cyan carbon is the modeled LTA 4 , and the CPK model is the bound GSH in which sulfur, carbon, nitrogen, and oxygen are in yellow, green, blue, and red, respectively.
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