Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein

We produced human leukotriene B(4) (LTB(4)) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB(4): (i) a low-affinity state (K(a)=7.8x10(8)M(-1)) that involves a receptor homodimer (BLT1.LTB(4))(2); we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (K(a)=1.3x10(10)M(-1)) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2). Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB(4), association with a G-protein and activation of Galpha. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1.LTB(4))(2):Galpha(i2)beta(1)gamma(2) pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB(4)-induced signal.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Leukotriene B<Subscript>4</Subscript> regulates proliferation and differentiation of cultured rat myoblasts via the BLT1 pathway

Skeletal muscle regeneration is a highly orchestrated process initiated by activation of adult muscle satellite cells. Upon muscle injury, the inflammatory process is always accompanied by muscle regeneration. Leukotriene B₄ is one of the essential inflammatory mediators. We isolated and cultured primary satellite cells. RT-PCR showed that myoblasts expressed mRNA for LTB₄ receptors BLT1 and BLT2, and LTB4 promoted myoblast proliferation and fusion. Quantitative real-time PCR and immunoblotting showed that LTB₄ treatment expedited the expression process of differentiation markers MyoD and M-cadherin. U-75302, a specific BLT1 inhibitor, but not LY2552833, a specific BLT2 inhibitor, blocked proliferation and differentiation of myoblasts induced by LTB₄, which implies the involvement of the BLT1 pathway. Overall, the data suggest that LTB₄ contributes to muscle regeneration by accelerating proliferation and differentiation of satellite cells. These authors contributed equally to this work.     

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C₄ (LTC₄) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC₄ and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC₄ and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC₄ precursor leukotriene A₄ (LTA₄) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC₄ synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC₄ secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC₄ and contribute to the insulin resistant state.     

Non-specific effects of leukotriene synthesis inhibitors on HeLa cell physiology

We examined the effects of various leukotriene synthesis inhibitors on calcium signalling in HeLa cells, before and after transfection with BLT₁. All of the inhibitors studied were found to reduce increases in intracellular calcium concentration induced by BLT₁, but also by an ionophore or activation of various G-protein coupled receptors, regardless of BLT₁ expression. In order to explore the mechanism of these apparently general effects we examined HeLa cell expression of leukotriene receptors and biosynthetic enzymes and found that the genes for key leukotriene synthesis enzymes and all of the leukotriene receptors were not expressed. Leukotrienes are involved in the pathology of a variety of cancers, and for HeLa cells leukotrienes have been reported to be important for aspects of the carcinogenic phenotype. We find that leukotriene synthesis inhibitors have non-specific effects, so careful controls are necessary to avoid interpreting non-specific effects as evidence for leukotriene involvement.     

The vitamin D receptor is necessary for 1alpha,25-dihydroxyvitamin D(3) to suppress experimental autoimmune encephalomyelitis in mice

The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3), suppresses autoimmune disease in several animal models including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The molecular mechanism of this immunosuppression is at present unknown. While 1alpha,25-dihydroxyvitamin D(3) is believed to function through a single vitamin D receptor, there are reports of other vitamin D receptors as well as a "nongenomic" mode of action. We have prepared the EAE model possessing the vitamin D receptor null mutation and determined if 1alpha,25-dihydroxyvitamin D(3) can suppress this disease in the absence of a functional vitamin D receptor. Vitamin D receptor null mice develop EAE although the incidence rate is one-half that of wild-type controls. The administration of 1alpha,25-dihydroxyvitamin D(3) had no significant effect on the incidence of EAE in the vitamin D receptor null mice, while it completely blocked EAE in the wild-type mice. We conclude that 1alpha,25-dihydroxyvitamin D(3) functions to suppress EAE through the well-known VDR and not through an undiscovered receptor or through a "nongenomic" mechanism.     

Asparagine362 is essential for zinc binding and catalysis in the peptidase reaction of Saccharomyces cerevisiae leukotriene A4 hydrolase

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A₄ hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.     

Altered leukotriene B4 metabolism in CYP4F18-deficient mice does not impact inflammation following renal ischemia

Inflammatory responses to infection and injury must be restrained and negatively regulated to minimize damage to host tissue. One proposed mechanism involves enzymatic inactivation of the pro-inflammatory mediator leukotriene B₄, but it is difficult to dissect the roles of various metabolic enzymes and pathways. A primary candidate for a regulatory pathway is omega oxidation of leukotriene B₄ in neutrophils, presumptively by CYP4F3A in humans and CYP4F18 in mice. This pathway generates ω, ω-1, and ω-2 hydroxylated products of leukotriene B₄, depending on species. We created mouse models targeting exons 8 and 9 of the Cyp4f18 allele that allows both conventional and conditional knockouts of Cyp4f18. Neutrophils from wild-type mice convert leukotriene B₄ to 19-hydroxy leukotriene B₄, and to a lesser extent 18-hydroxy leukotriene B₄, whereas these products were not detected in neutrophils from conventional Cyp4f18 knockouts. A mouse model of renal ischemia–reperfusion injury was used to investigate the consequences of loss of CYP4F18 in vivo. There were no significant changes in infiltration of neutrophils and other leukocytes into kidney tissue as determined by flow cytometry and immunohistochemistry, or renal injury as assessed by histological scoring and measurement of blood urea nitrogen. It is concluded that CYP4F18 is necessary for omega oxidation of leukotriene B₄ in neutrophils, and is not compensated by other CYP enzymes, but loss of this metabolic pathway is not sufficient to impact inflammation and injury following renal ischemia–reperfusion in mice.     

Regulation of the eicosanoid pathway by tumour necrosis factor alpha and leukotriene D4 in intestinal epithelial cells

In this study the mRNA and protein levels of the key enzymes involved in eicosanoid biosynthesis and the cysteinyl leukotriene receptors (CysLT₁R and CysLT₂R) have been analysed in non-transformed intestinal epithelial and colon cancer cell lines. Our results revealed that tumour necrosis factor alpha (TNF-α), and leukotriene D₄ (LTD₄), which are inflammatory mediators implicated in carcinogenesis, stimulated an increase of cyclooxygenase-2 (COX-2), in non-transformed epithelial cells, and 5-lipoxygenase (5-LO) in both non-transformed and cancer cell lines. Furthermore, these mediators also stimulated an up-regulation of LTC₄ synthase in cancer cells as well as non-transformed cells. We also observed an endogenous production of CysLTs in these cells. TNF-α and LTD₄, to a lesser extent, up-regulate the CysLT₁R levels. Interestingly, TNF-α also reduced CysLT₂R expression in cancer cells. Our results demonstrate that inflammatory mediators can cause intestinal epithelial cells to up-regulate the expression of enzymes needed for the biosynthesis of eicosanoids, including the cysteinyl leukotrienes, as well as the signal transducing proteins, the CysLT receptors, thus providing important mechanisms for both maintaining inflammation and for tumour progression.     

Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1

We produced the human leukotriene B(4) (LTB(4)) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% alpha-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB(4) with K(a)=7.8(+/-0.2)x10(8)M(-1) and a stoichiometric ratio of 0.98(+/-0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB(4). We report evidence that both partners, LTB(4) and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB(4) conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.     

Suppression of murine experimental autoimmune encephalomyelitis by interleukin-2 receptor targeted fusion toxin, DAB389IL-2

Previously we have shown that DAB₃₈₉IL-2, a recombinant fusion toxin targeting IL-2R bearing cells, suppressed disease in the rat experimental autoimmune encephalomyelitis (EAE) model of acute multiple sclerosis (MS). Our present study demonstrates that DAB₃₈₉IL-2 can also effectively suppress acute (A)-EAE, relapsing (R)-EAE and chronic (C)-EAE in mouse demyelinating models. DAB₃₈₉IL-2 significantly suppressed mitogenic proliferation of spleen cells while mutant fusion proteins DA_glu53B₃₈₉IL-2 and DAB₃₈₉IL-2_8-10 did not. EAE was successfully suppressed when DAB₃₈₉IL-2 was administered in various regimens between days 1 and 15 post immunization in all three models. CD4⁺IL-2R⁺ cells were reduced in the spleen but not in the lymph nodes of DAB₃₈₉IL-2-treated mice during A-EAE while the number of CD8⁺ cells was unchanged. DAB₃₈₉IL-2 also significantly reduced the number of CD4⁺, CD8⁺, CD25⁺, TCRγδ⁺ phenotype and CD11b⁺ macrophages/microglia within spinal cord lesions. These data strongly suggest that DAB₃₈₉IL-2 specifically targeted myelin protein-activated CD4⁺ T cells and strengthens the argument for the use of DAB₃₈₉IL-2 in treatment strategies for MS.     

Effect of leukotriene C4 exposure on ciliated cells of the nasal mucosa

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10⁻⁶M to 10⁻⁹M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10⁻⁸M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a γ-glutamyl transpeptidase (γ-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by γ-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.     

拟南芥抗凋亡突变体fbr136的分离鉴定与分析

植物细胞程序性死亡(programmed cell death,PCD)在植物的生长发育进程以及防御生物与非生物胁迫的过程中具有重要的作用.Fumonisin B1(FB1)是一种真菌毒素,是鞘脂生物合成途径中关键酶神经酰胺合酶(ceramide synthase)的竞争性抑制剂.FB1在动植物细胞中均能够诱导PCD.为了探索植物PCD的机制,通过筛选拟南芥抗FB1的突变体,分离鉴定了11个,fumonisin B1 resistant (fbr)突变体.遗传分析表明,这些突变体分别是由9个相同或者不同的遗传座位突变造成的.对其中一个代表性的突变体fbr136进行了详细的表型分析和初步遗传定位.fbr136对其他PCD诱导剂,例如H2O2或paraquat也表现出一定的抗性或耐受性,而且在fbr136突变体中FB1不能正常诱导PR1基因的表达,说明fbr136突变体PCD的发生可能受到阻碍.硝基四唑(Nitroblue tetrazolium,NBY)染色表明,FBl处理fbr136突变体后产生和积累活性氧(reactive oxygenspecies)比野生型植物显著降低,暗示其抗凋亡表型可能与活性氧的产生有关.推测FBR136可能是FB1在诱导PCD过程中,从鞘脂含量变化到活性氧积累变化这一途径的一个重要的调控因子.fbr136被定位于染色体Ⅲ上,与以往鉴定的抗FB1突变基因的定位都不同,因此,FBR136可能是FB1诱导PCD信号途径中的一个新基因.     

Is the expression of kinin B1 receptor related to intrahepatic vascular response?

Bradykinin elicits an intrahepatic vascular response (IHVR) mediated by the constitutive B₂ receptor (B₂R). The biological effects of kinins may also be mediated by the inducible B₁ receptor (B₁R). Aim: To verify if the hepatic B₁R expression modulates IHVR to kinins. Method: We evaluated the ability of bradykinin and B₁R agonists to elicit an IHVR in normal rats and in those submitted to acute or chronic inflammatory stimuli, fibrosis, cirrhosis, or hepatic regeneration. Results: Bradykinin-induced IHVR was similar in all groups. B₁R agonists did not elicit in any of them either a hypertensive or a hypotensive response. B₁ receptor induction was observed in all experimental groups (Western blot), except for the acute inflammatory group. Conclusion: B₁R hepatic expression did not modulate IHVR to kinins.     

Attenuation of ischemic efflux of endogenous amino acids by the novel 5-HT1A/5-HT2 receptor ligand adatanserin

Using sodium azide (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia in the hippocampal slice, we have evaluated the effects of the novel 5-HT(1A) partial agonist/5-HT(2) receptor antagonist adatanserin and the 5-HT(1A) receptor agonist BAYx3702 on the efflux of endogenous glutamate, aspartate and GABA. BAYx3702 (10-1000 nM) produced a significant (P<0.05) dose-related attenuation of ischemic efflux of both glutamate and GABA with maximum decrease being observed at 100 nM (73 and 69%, respectively). This attenuation was completely reversed by the addition of the 5-HT(1A) antagonist, WAY-100635 (100 nM). Similarly, adatanserin (10-1000 nM) produced a significant (P<0.05) dose-related attenuation in glutamate and GABA efflux with a maximum of 72 and 81% at 100 nM, respectively. This effect was completely reversed by the 5-HT(2A/C) receptor agonist, DOI but unaffected by WAY-100635. The 5-HT(2A) receptor antagonist MDL-100907 produced a comparable attenuation of glutamate when compared to adatanserin, while the 5-HT(2C) receptor antagonist, SB-206553, had no effect on ischemic efflux. None of these compounds significantly altered aspartate efflux from this preparation. In conclusion, the 5-HT(1A) receptor partial agonist 5-HT(2) receptor antagonist, adatanserin is able to attenuate ischemic amino acid efflux in a comparable manner to the full 5-HT(1A) agonist BAYx3702. However, in contrast to BAYx3702, adatanserin appears to produce it effects via blockade of the 5-HT(2A) receptor. This suggests that adatanserin may be an effective neuroprotectant, as has been previously demonstrated for full 5-HT(1A) receptor agonists such as BAYx3702.     

Prostaglandin E1 or E2 inhibits an oxytocin-induced premature luteolysis in ewes when oxytocin is given early in the estrous cycle

The objective of this study was to determine whether PGE₁ or PGE₂ prevents a premature luteolysis when oxytocin is given on Days 1 to 6 of the ovine estrous cycle. Oxytocin given into the jugular vein every 8 hours on Days 1 to 6 postestrus in ewes decreased (P ≤ 0.05) luteal weights on Day 8 postestrus. Plasma progesterone differed (P ≤ 0.05) among the treatment groups; toward the end of the experimental period, concentrations of circulating progesterone in the oxytocin-only treatment group decreased (P ≤ 0.05) when compared with the other treatment groups. Plasma progesterone concentrations in ewes receiving PGE₁ or PGE₁ + oxytocin were greater (P ≤ 0.05) than in vehicle controls or in ewes receiving PGE₂ or PGE₂ + oxytocin and was greater (P ≤ 0.05) in all treatment groups receiving PGE₁ or PGE₂ than in ewes treated only with oxytocin. Chronic intrauterine treatment with PGE₁ or PGE₂ also prevented (P ≤ 0.05) oxytocin decreases in luteal unoccupied and occupied LH receptors on Day 8 postestrus. Oxytocin given alone on Days 1 to 6 postestrus in ewes advanced (P ≤ 0.05) increases in PGF_2α in inferior vena cava or uterine venous blood. PGE₁ or PGE₂ given alone did not affect (P ≥ 0.05) concentrations of PGF_2α in inferior vena cava and uterine venous blood when compared with vehicle controls or oxytocin-induced PGF_2α increases (P ≤ 0.05) in inferior vena cava or uterine venous blood. We concluded that PGE₁ or PGE₂ prevented oxytocin-induced premature luteolysis by preventing a loss of luteal unoccupied and occupied LH receptors.     

Discovery, design and synthesis of the first reported potent and selective sphingosine-1-phosphate 4 (S1P4) receptor antagonists

Selective S1P₄ receptor antagonists could be novel therapeutic agents for the treatment of influenza infection in addition to serving as a useful tool for understanding S1P₄ receptor biological functions. 5-(2,5-Dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide was identified from screening the Molecular Libraries-Small Molecule Repository (MLSMR) collection and selected as a promising S1P₄ antagonist hit with moderate in vitro potency and high selectivity against the other family receptor subtypes (S1P_1-3,5). Rational chemical modifications of the hit allowed the disclosure of the first reported highly selective S1P₄ antagonists with low nanomolar activity and adequate physicochemical properties suitable for further lead-optimization studies.     

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

Crystal structure of leukotriene A4 hydrolase in complex with kelatorphan, implications for design of zinc metallopeptidase inhibitors

Leukotriene A₄ hydrolase (LTA4H) is a key enzyme in the inflammatory process of mammals. It is an epoxide hydrolase and an aminopeptidase of the M1 family of the MA clan of Zn-metallopeptidases. We have solved the crystal structure of LTA4H in complex with N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine, a potent inhibitor of several Zn-metalloenzymes, both endopeptidases and aminopeptidases. The inhibitor binds along the sequence signature for M1 aminopeptidases, GXMEN. It exhibits bidentate chelation of the catalytic zinc and binds to LTA4H’s enzymatically essential carboxylate recognition site. The structure gives clues to the binding of this inhibitor to related enzymes and thereby identifies residues of their S1′ sub sites as well as strategies for design of inhibitors.