1. Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan
2. Department of Medicine, Harvard Medical School and Division of Rheumatology, Immunology, and Allergy, Brigham Women's Hospital, Boston, Massachusetts 02115, USA
3. These authors contributed equally to this work.

Correspondence to: K. Frank Austen²Masashi Miyano¹ Correspondence and requests for materials should be addressed to M.M. (Email: miyano@spring8.or.jp) or K.F.A. (Email: fausten@rics.bwh.harvard.edu).

Abstract

The cysteinyl leukotrienes, namely leukotriene (LT)C₄ and its metabolites LTD₄ and LTE₄, the components of slow-reacting substance of anaphylaxis^{1, 2}, are lipid mediators of smooth muscle constriction^{3, 4, 5} and inflammation^{6, 7}, particularly implicated in bronchial asthma^{8, 9}. LTC₄ synthase (LTC₄S), the pivotal enzyme for the biosynthesis of LTC₄ (ref. 10), is an 18-kDa integral nuclear membrane protein^{11, 12} that belongs to a superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism that includes 5-lipoxygenase-activating protein, microsomal glutathione S-transferases (MGSTs), and microsomal prostaglandin E synthase 1 (ref. 13). LTC₄S conjugates glutathione to LTA₄, the endogenous substrate derived from arachidonic acid through the 5-lipoxygenase pathway¹⁴. In contrast with MGST2 and MGST3 (refs 15, 16), LTC₄S does not conjugate glutathione to xenobiotics¹⁷. Here we show the atomic structure of human LTC₄S in a complex with glutathione at 3.3 Å resolution by X-ray crystallography and provide insights into the high substrate specificity for glutathione and LTA₄ that distinguishes LTC₄S from other MGSTs. The LTC₄S monomer has four transmembrane -helices and forms a threefold symmetric trimer as a unit with functional domains across each interface. Glutathione resides in a U-shaped conformation within an interface between adjacent monomers, and this binding is stabilized by a loop structure at the top of the interface. LTA₄ would fit into the interface so that Arg 104 of one monomer activates glutathione to provide the thiolate anion that attacks C6 of LTA₄ to form a thioether bond, and Arg 31 in the neighbouring monomer donates a proton to form a hydroxyl group at C5, resulting in 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC₄). These findings provide a structural basis for the development of LTC₄S inhibitors for a proinflammatory pathway mediated by three cysteinyl leukotriene ligands whose stability and potency are different and by multiple cysteinyl leukotriene receptors whose functions may be non-redundant.

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