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

Abstract

The cysteinyl leukotrienes, namely leukotriene (LT)C4 and its metabolites LTD4 and LTE4, the components of slow-reacting substance of anaphylaxis, are lipid mediators of smooth muscle constriction and inflammation, particularly implicated in bronchial asthma. LTC4 synthase (LTC4S), the pivotal enzyme for the biosynthesis of LTC4 (ref. 10), is an 18-kDa integral nuclear membrane protein 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 pathway. In contrast with MGST2 and MGST3 (refs 15, 16), LTC4S does not conjugate glutathione to xenobiotics. Here we show the atomic structure of human LTC4S in a complex with glutathione at 3.3 A 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 alpha-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.

Structural basis for induced formation of the inflammatory mediator prostaglandin E 2

“…The subunits of MPGES1 form a homotrimer ( Fig. 1) in a similar way as for the other structurally characterized MAPEG members, MGST1 (17), FLAP (18), and LTC4S (19,20) and in agreement with earlier low resolution and hydrodynamic data on MPGES1 (5). Our result thus supports the suggestion that a trimeric arrangement is common to all MAPEG proteins (21).…”

“…These regions were U-shaped (Fig. 3B) similar to what has been observed for GSH in LTC4S (19,20) but located deeper and at a different angle. In the vicinity of the GSH binding pocket a number of conserved residues are found that are involved in GSH binding (Fig.…”

“…Hydrogen/deuterium exchange dynamics of the closely related MGST1 supports dynamic f lexibility in helix 1 (22) that is decreased when a hydrophobic ligand is present (23). Furthermore, in the structures of LTC4 synthase a detergent molecule occupies the V-shaped cleft between subunits, which was proposed to be the substrate binding site (19,20). To generate a model (24) of an open structure for MPGES1, we used LTC4S as a template (Fig.…”

“…In LTC4S an opening between helix 1 in one subunit and helix 4 in a neighboring subunit forms a V-shaped cleft that allows access to GSH from the interior of the membrane (19,20). This represents a natural entry point for labile hydrophobic substrates, such as PGH 2 and LTA 4 that, by way of membrane entry, are protected from hydrolytic degradation.…”

The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides used in membrane protein crystallization

“…8 n-Alkyl-b-D-maltosides (C n G 2 ), which are in the focus of the present study, have been used in many cases as solubilizing agents to achieve membrane protein crystallization. Representative examples of a successful application of C 12 G 2 are photosystem I (PSI) 9 and photosystem II (PSII) [10][11][12] as well as the cytochrome (cyt) b 6 f complex 13 of oxygenic photosynthesis, a bacterial multidrug efflux transporter, 14 a cyt c quinol dehydrogenase, 15 a human leukotriene C 4 synthase, 16,17 and the rotor of V-type Na + -ATPase. 18 The homologous detergent C 11 G 2 was used in the case of the yeast cyt bc 1 complex, 19,20 C 10 G 2 for a glutamate transporter, 21 and C 8 G 2 for a chloride channel.…”