FEBS Letters 584 (2010) 3446–3451 journal homepage: www.FEBSLetters.org Crystal structure of leukotriene A4 hydrolase in complex with kelatorphan, implications for design of zinc metallopeptidase inhibitors q Fredrik Tholander a,1, Bernard-Pierre Roques c, Marie-Claude Fournié-Zaluski d, Marjolein M.G.M. Thunnissen b, Jesper Z. Haeggström a,* a Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institute, S-171 77 Stockholm, Sweden b Department of Molecular Biophysics, Kemicentrum, Lund University, S-221 00 Lund, Sweden c Départment de Pharmacochimie Moléculaire et Structurale, 4 Avenue de l’Observatoire, 75270 Paris Cedex 06, France d Pharmaleads, 11 rue Watt, 7513 Paris, France article info abstract Article history: Leukotriene A4 hydrolase (LTA4H) is a key enzyme in the inflammatory process of mammals. It is an Received 1 June 2010 epoxide hydrolase and an aminopeptidase of the M1 family of the MA clan of Zn-metallopeptidases. Revised 24 June 2010 We have solved the crystal structure of LTA4H in complex with N-[3(R)-[(hydroxyamino)carbonyl]- Accepted 25 June 2010 2-benzyl-1-oxopropyl]-L-alanine, a potent inhibitor of several Zn-metalloenzymes, both endopep- Available online 6 July 2010 tidases and aminopeptidases. The inhibitor binds along the sequence signature for M1 aminopepti- Edited by Christian Griesinger dases, 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 S10 sub sites as well as strategies Keywords: Leukotriene for design of inhibitors. Leukotriene B4 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. LTA4 hydrolase Aminopeptidase Cardiovascular Inflammation 1. Introduction to participate in the host-defense against infection [3], to regulate lipid metabolism [4] and to be a key-mediator of the lethal effect of Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc metal- PAF-induced systemic shock [5,6]. Elevated levels of LTB4 have loenzyme possessing an aminopeptidase as well as an epoxide been detected in afflicted tissues of several inflammatory diseases, hydrolase activity. Whereas the former activity is yet of unknown e.g., rheumatoid arthritis [7], inflammatory bowel disease [8] and physiological relevance the latter catalyzes the formation of the psoriasis [9]. It has recently been demonstrated that LTA4H is potent chemotaxin leukotriene B4 (LTB4), a substance which is fun- upregulated in atherosclerotic plaques of humans thus suggesting damental to the inflammatory response of mammals and possibly a role for LTB4 in cardiovascular disease [10]. Furthermore, recent all vertebrates. Even though the immediate effect of LTB4 is neutro- reports have shown that LTB4 also mediates recruitment of certain phil recruitment, which is elicited already at nanomolar concentra- T-cells to the site of inflammation, thereby identifying LTB4 as a tions [1], LTB4 is also known to modulate the immune response [2], link between the innate and adaptive immune response [11–13]. Since LTB4 is undoubtedly of pathological relevance it is partic- ularly interesting to control its biosynthesis as a means to treat dif- Abbreviations: p-NAn, para-nitroaniline; p-NA, para-nitroanilide; LTA4, leuko- ferent inflammatory disorders. To this end, the development and triene A4; LTA4H, Leukotriene A4 hydrolase; SDS–PAGE, sodium dodecyl polyacryl- analysis of efficient inhibitors targeted against LTA4H is highly amide gel electrophoresis; HPLC, high performance liquid chromatography; interesting. Here we report the crystal structure of LTA4H in com- kelatorphan, N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine q Atomic coordinates and structure factors are available at the Protein Data Bank plex with N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopro- (http://www.rcsb.org/) with PDBID 3B7U. pyl]-L-alanine (kelatorphan), a di-peptide related substance in * Corresponding author. Fax: +46 8 7360439. which the a-amino group has been replaced by a bidentate me- E-mail addresses: [email protected] (F. Tholander), jesper.haegg- tal-chelating hydroxamic acid moiety. [email protected] (J.Z. Haeggström). Kelatorphan was originally designed to inhibit the endopepti- 1 Present address: Department of Molecular Biology and Functional Genomics, Stockholm University, 106 91 Stockholm, Sweden. dase neprilysin (Ki 2 nM) but was subsequently found to be potent 0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.06.044 F. Tholander et al. / FEBS Letters 584 (2010) 3446–3451 3447 also towards human aminopeptidase N (APN) (Ki 0.7 lM) and acetic acid (10%). Metabolites were isolated by solid phase extrac- LTA4H (Ki of 5 nM), even though it does not contain the free amino tion on Chromabond C18 columns. Samples were loaded on the group typically required for recognition by aminopeptidases [14– columns and washed with 1 ml 25% methanol followed by extrac- 16]. Here we report the crystal structure of LTA4H in complex with tion with 250 ll 100% methanol. Extracted samples were diluted kelatorphan, which reveals a binding arrangement relying on a with 250 ll water and subsequently analyzed by high performance conserved sequence motif (GXMEN of M1 aminopeptidases), liquid chromatography with a Waters Nova-Pak C18 column and strong zinc chelation and binding to LTA4H’s carboxylate recogni- isocratic elution with methanol/acetonitrile/water/acetic acid tion site. Since binding of kelatorphan relies on conserved struc- (30:30:40:0.01 by volume) at a flow rate of 1.2 ml/min. Metabo- tural elements it is possible to extrapolate our findings to other lites were detected by their UV absorbance at 270 nm and quanti- homologous Zn metallo enzymes. fied using Chromatography Station for Windows version 1.7 computer software. Calculations were based on peak area mea- 2. Materials and methods surements and the known extinction coefficients for the internal À1 À1 À1 standard PGB2 (30 000 M Â cm )aswellasLTB4 (50 000 M Â 2.1. Materials cmÀ1). Leukotriene A4 (LTA4) methyl ester was purchased from BIO- 2.5. Determination of kinetic parameters MOL Res. Lab, Plymouth Meeting, PA, USA and saponified in tetra- hydrofuran with 1 M LiOH (6% v/v) for 48 h at 4 °C. Bradford Data obtained from the aminopeptidase inhibition assay was protein dye-reagent was from Bio-Rad (Bio-Rad Laboratories AB, analyzed using the Dynafit program [21]. The program numerically Sweden) and other chemicals were from Sigma (Sigma–Aldrich fits various models for inhibition to the data and performs model Sweden AB, Sweden). Kelatorphan was synthesized as described discrimination analysis. Ki values from the model obtaining the [17,18]. highest statistical significance are reported and standard errors are the ones calculated by the software. 2.2. Protein expression and purification For determination of IC50 values, a standard four parameter Hill equation (with parameters for top and bottom plateaus set to con- Expression of (His)6-tagged protein in Escherichia coli and sub- stant values) was fitted to the data and the model-to-data fit ana- sequent purification with Ni-affinity chromatography followed by lyzed using the Solverstat software [22]. anion exchange chromatography was performed as previously de- scribed [16,19]. The final anion exchange step was only performed 2.6. Crystallization on protein preparations used for crystallization. The protein purity was assessed by sodium dodecyl polyacrylamide gel electrophore- Crystals of LTA4H complexed with kelatorphan were obtained sis (SDS–PAGE) using 10–15% gradient gels on a Pharmacia phast by co-crystallization of enzyme and inhibitor using the liquid–li- system. Protein concentration was determined according to the quid diffusion method described previously [19]. Briefly, 5 llof Bradford method using bovine serum albumin as standard. To ver- precipitation solution (28% (v/v) PEG8000, 50 mM sodium acetate, ify the functional integrity of the protein, the tri-peptidase activity 0.1 M imidazole buffer, pH 6.8, and 5 mM YbCl3), was injected into was confirmed with a recently described novel methodology utiliz- the bottom of a melting point capillary and an equal volume of ing competing substrates [20]. LTA4H (5 mg/ml) in 10 mM Tris–HCl, pH 7.5, containing 1 mM kelatorphan, was layered on top. 2.3. Aminopeptidase inhibition assays 2.7. Diffraction data collection Five sets of initial velocity measurements were performed. Within a set, substrate concentration was varied while concentra- For data collection, crystals were soaked in 14% (w/v) PEG8000, tion of inhibitor was kept constant. Inhibitor concentration was 25 mM sodium acetate, 50 mM imidazole buffer, pH 6.8, 2.5 mM varied between sets. The complete experiment was repeated 3 YbCl3 and 25% (v/v) glycerol. Data was collected at the beamline times. I711 of Max-Lab, Lund, Sweden. During data collection the crystal Aliquots (50 ll) of equilibrated solutions containing 1 lg pro- was kept under a stream of liquid nitrogen. A complete set of data tein, 500 mM KCl, 250 mM Tris–HCl pH 7.5 and various amounts was collected from one single crystal. of inhibitor were added to the wells of a microtiter plate. Reactions were initiated by the addition of 200 ll of ala-p-NA of various con- 2.8. Data processing and refinement centrations. The amount of para-nitroaniline (p-NAn) formation was monitored for 15 min in a MCC/340 multiscan spectrophotom- Diffraction data were processed with Mosflm [23] and programs eter as increase in absorbance at 405 nm. To correct for spontane- of the CCP4 program suit [24]. As a starting model for the refine- ous hydrolysis of ala-p-NA, absorbance of incubations without ment the crystal structure of [E271Q]LTA4H was used (PDB ID enzyme was subtracted. A molar response factor of 8065 MÀ1 1H19).