Crystal Structure of Inhibitor-Bound Human 5-Lipoxygenase-Activating Protein

Ferguson, Andrew D.; McKeever, Brian; Xu, Suoyu; Wisniewski, Douglas; Miller, Douglas K.; Yamin, Ting-Ting; Spencer, Robert H.; Chu, Lin; Ujjainwalla, Feroze; Cunningham, Barry R.; Evans, Jilly F.; Becker, Joseph W.

doi:10.1126/science.1144346

Cited by 206 publications

(171 citation statements)

References 26 publications

(7 reference statements)

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“…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).…”

Section: Resultssupporting

confidence: 76%

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

Jegerschöld

Pawelzik²,

Purhonen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

141

204

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Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH 2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH 2 to PGE 2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.electron crystallography ͉ inflammation ͉ MAPEG ͉ membrane protein M icrosomal prostaglandin E synthase 1 (MPGES1) is the key enzyme in pathology related production of PGE 2 from cyclooxygenase (Cox) derived PGH 2 (1). The protein is a member of the MAPEG protein family, which includes 5-lipoxygenase activating protein (FLAP), leukotriene C 4 synthase (LTC4S), microsomal glutathione transferase (MGST)1, MGST2, and MGST3 (2, 3). MPGES1 is the most efficient PGES known and catalyzes the oxidoreduction of prostaglandin endoperoxide H 2 into PGE 2 with an apparent k cat /K m of 310 mM Ϫ1 s Ϫ1 [supporting information (SI) Fig. S1]. The enzyme equally well catalyses the oxidoreduction of endocannabinoids into prostaglandin glycerol esters (4) and PGG 2 into 15-hydroperoxy-PGE 2 (5). In addition, the enzyme confers low glutathione transferase and glutathione-dependent peroxidase activities (5). The biological significance of the latter activities remains unclear but is thought to reflect the close evolutionary distance to MGST1.MPGES1 protein expression levels are in most cases low, and proinflammatory stimuli induce its cellular expression and activity, which is prevented by corticosteroids (1, 6-8). The predominant source of PGH 2 seems derived from Cox-2, although Cox-1 may also contribute (9). Studies, mainly from disruption of the MPGES1 gene in mice, indicate key roles for MPGES1-generated PGE 2 in pathological conditions such as chronic inflammation, pain, fever, anorexia, atherosclerosis, stroke and tumorigenesis (10). Recently, a role for MPGES1 in regulating neonatal respiration was described in ref. 11. MPGES1 has been shown to be overexpressed in rheu...

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Section: Resultssupporting

confidence: 76%

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

Jegerschöld

Pawelzik²,

Purhonen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

141

204

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“…Secondly, the combination of mutagenesis and structure determination shows a partial overlap between the AA and inhibitor binding sites of FLAP (16,23). These sites are positioned within the membrane, ideally situated to capture laterally diffusing AA generated exogenously to the complex; FLAP-associated AA could be made available to 5-LO (16,23). The structural basis of how FLAP brings 5-LO into apposition with AA remains a key question, as does the site(s) at which 5-LO interacts with FLAP.…”

Section: Discussionmentioning

confidence: 99%

“…Several considerations made antibody (Ab)-based Fluorescence Lifetime Imaging Microscopy (FLIM) the approach of choice to image the interaction between 5-LO and FLAP. First, based on its crystal structure (16) the N-and C-termini of FLAP are on the opposite side of the membrane from 5-LO. Second, the placement of fusion proteins on 5-LO must be on the N-terminal of the enzyme to preserve catalytic function.…”

Section: -Lo and Flapmentioning

confidence: 99%

The nuclear membrane organization of leukotriene synthesis

Mandal

Jones

Bair

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.inflammation ͉ multiprotein complex ͉ 5-lipoxygenase ͉ 5-lipoxygenase-activating protein L eukotrienes (LTs) are lipid signaling molecules derived from arachidonic acid (AA) that initiate and amplify innate and adaptive immune responses by regulating the recruitment and activation of leukocytes in inflamed tissues (1-3). Cells employ multiple mechanisms to prevent the inappropriate onset of pro-inflammatory signaling while requiring tightly coupled processes to trigger the generation of pro-inflammatory signaling molecules. The interplay of these processes is epitomized by the synthesis of LTB 4 and LTC 4 . In unstimulated myeloid cells, including mast cells, LT formation is held in abeyance by the cytosolic compartmentalization of cytosolic phospholipase A 2 (cPLA 2 ) (4, 5) and the cytosolic/nucleoplasmic localization of 5-lipoxygenase (5-LO) (6). LT formation is initiated by translocation of cPLA 2 to the Golgi and ER/nuclear envelope to release AA (4, 5). In parallel, 5-LO targets to the inner and outer nuclear membranes to initiate LT synthesis by converting AA to 5-HPETE and LTA 4 , a process that requires the integral nuclear envelope protein 5-lipoxygenase-activating protein (FLAP) (7-9). Although we have shown that LTC 4 synthase and FLAP constitutively interact on the nuclear envelope (10), how or whether 5-LO interacts with these proteins on the nuclear envelope in response to cell signaling is unknown.Understanding how cells couple the reorganization of the LT biosynthetic enzymes to efficient AA utilization is central to understanding the signal transduction pathways that control the synthesis of bioactive lipids derived from AA. Scaffold/docking proteins can localize components of biochemical reactions at membrane interfaces; examples include protein kinase A anchoring proteins (11) and the integral membrane proteins caveolins 1-3 (12). The dependence of cellular LT synthesis on FLAP (7, 8) and its membrane localization (9) make it a conceptually appealing candidate for a 5-LO scaffold. However, no interaction of 5-LO with any membrane protein has been detected.A closely related question is: how do different combinations of extracellular signals lead to LT generation? For example, in mast cells, the engage...

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“…23 There is one reported case of a membrane protein being extracted, purified, and crystallized in another foscholine detergent, FC14, the E. coli mechanoselective channel McsC. 24,25 OmpF overexpression during recombinant membrane protein expression in E. coli Recenly, the first crystal structure of a human membrane protein expressed in E. coli 26,27 has renewed interest in E. coli-based expression of mammalian membrane proteins. 28,29 OmpF contamination is a major issue that needs to be seriously considered.…”

Section: Crystal Packing Of Ompf Prepared In Fc12mentioning

confidence: 99%

Structures of the OmpF porin crystallized in the presence of foscholine‐12

et al. 2010

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The endogenous Escherichia coli porin OmpF was crystallized as an accidental byproduct of our efforts to express, purify, and crystallize the E. coli integral membrane protein KdpD in the presence of foscholine-12 (FC12). FC12 is widely used in membrane protein studies, but no crystal structure of a protein that was both purified and crystallized with this detergent has been reported in the Protein Data Bank. Crystallization screening for KdpD yielded two different crystals of contaminating protein OmpF. Here, we report two OmpF structures, the first membrane protein crystal structures for which extraction, purification, and crystallization were done exclusively with FC12. The first structure was refined in space group P21 with cell parameters a 5 136.7 Å , b 5 210.5 Å , c 5 137 Å , and b 5 100.5°, and the resolution of 3.8 Å . The second structure was solved at the resolution of 4.4 Å and was refined in the P321 space group, with unit cell parameters a 5 215.5 Å , b 5 215.5 Å , c 5 137.5 Å , and c 5 120°. Both crystal forms show novel crystal packing, in which the building block is a tetrahedral arrangement of four trimers. Additionally, we discuss the use of FC12 for membrane protein crystallization and structure determination, as well as the problem of the OmpF contamination for membrane proteins overexpressed in E. coli.

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Crystal Structure of Inhibitor-Bound Human 5-Lipoxygenase-Activating Protein

Cited by 206 publications

References 26 publications

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

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

The nuclear membrane organization of leukotriene synthesis

Structures of the OmpF porin crystallized in the presence of foscholine‐12

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Crystal Structure of Inhibitor-Bound Human 5-Lipoxygenase-Activating Protein

Cited by 206 publications

References 26 publications

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

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

The nuclear membrane organization of leukotriene synthesis

Structures of the OmpF porin crystallized in the presence of foscholine‐12

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Product

Resources

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

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