Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis

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Abstract

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis1,2, are lipid mediators of smooth muscle constriction3,4,5 and inflammation6,7, particularly implicated in bronchial asthma8,9. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein11,12 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 α-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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Figure 1: Structure of LTC 4 S.
Figure 2: GSH-binding site at the interface of monomers C and A.
Figure 3: The V-shaped valley as the putative LTA 4 -binding site.

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Acknowledgements

We thank T. Tsurumura and N. Takahashi for their technical assistance. This work was supported by Tanpaku3000 of MEXT (to M.M.), RIKEN Matching fund (to H.A. and M.M.) and National Institutes of Health grants (to Y.K., B.K.L. and K.F.A.).

Author Contributions H.A., Y.K. and D.I. contributed equally to this work. Y.K. overexpressed and purified the human LTC4S. H.A. optimized the enzyme purification methods. H.A., D.I. and T.S. crystallized the enzyme. D.I. prepared the SeMet protein. H.A. and M.M. solved the structure. B.K.L. performed the LTC4S assay. H.A., Y.K., K.F.A. and M.M. guided the work and prepared the manuscript. All authors discussed the results and commented on the manuscript.

Coordinates and structure factors have been deposited in the Protein Data Bank with the accession number 2PNO.

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Correspondence to K. Frank Austen or Masashi Miyano.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Figures S1-S3 with Legends and Table S1 with crystallographic statistics. (PDF 3422 kb)

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