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Structure of Human Neutrophil Elastase in Complex with a Peptide

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Structure of Human Neutrophil Elastase in Complex with a Peptide Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7-11, January 1989 Biochemistry Structure of human neutrophil elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84-A resolution (crystallography/l-lactams/neutrophil granules) MANUEL A. NAVIA*t, BRIAN M. MCKEEVER*, JAMES P. SPRINGER*, TSAU-YEN LINt, HOLLIS R. WILLIAMSt, EUGENE M. FLUDER§, CONRAD P. DORN¶, AND KARST HOOGSTEEN* Merck Sharp & Dohme Research Laboratories, Departments of *Biophysical Chemistry, tEnzymology, §Scientific Programming, and 1Membrane and Arthritis Research, Rahway, NJ 07065 Communicated by David R. Davies, August 12, 1988 (receivedfor review January 29, 1988) ABSTRACT Human neutrophil elastase (HNE) has been the molecule, although coordinates have not been available implicated as a major contributor to tissue destruction in for a quantitative comparison. various disease states, including emphysema. The structure of Due to the clinical importance ofHNE, numerous peptide- HNE, at neutral pH, in complex with methoxysuccinyl- based as well as nonpeptidyl inhibitors of this enzyme and of Ala-Ala-Pro-Ala chloromethyl ketone (MSACK), has been the related PPE have been examined (9, 14). Peptide chlo- solved and refmed to anR factor of 16.4% at 1.84-A resolution. romethyl ketones such as MSACK have been shown to be Results are consistent with the currently accepted mechanism effective in vivo in arresting the development of experimen- of peptide chloromethyl ketone inhibition of serine proteases, tally induced emphysema in animal models of the disease in that MSACK cross-links the catalytic residues His-57 and (15). They have also served as a standard of comparison for newly developed inhibitors, even though they present serious Ser-195. The structure of the HNE-MSACK complex is com- side effects (16). The structure of the inactivated HNE- pared with that of porcine pancreatic elastase in complex with MSACK complex, in defining peptide binding to the enzyme, L-647,957, a fi-lactam inhibitor of both elastases. The distri- should be useful in the development of safer inhibitors. bution of positively charged residues on HNE is highiy asym- Recently, we reported the three-dimensional structure of metric and may play a role in its specific association with the L-647,957 (3-acetoxymethyl-7a-chloro-3-cephem-4-carboxy- underlying negatively charged proteoglycan matrix of the late-1,1-dioxide tert-butyl ester), a f-lactam HNE inhibitor, neutrophil granules in which the enzyme is stored. in complex with PPE (17). By comparing the structures ofthe two complexes, we can extend with confidence the PPE Human neutrophil elastase (HNE) is capable of digesting the ,3-lactam inhibition results to HNE, the actual drug target of underlying elastin structure of the alveolar walls of the lung interest. (1). Normally, a, protease inhibitor, a naturally occurring protein present in the lung (2) acts to suppress HNE-mediated damage. However, if a, protease inhibitor is present in MATERIALS AND METHODS unusually low amounts or if it is nonfunctional, either HNE derived from human purulent sputum was purchased because of an underlying genetic defect (3) or due to smoking from Elastin Products, Saint Louis. Details of the final (4), the development of emphysema can follow. Direct a, purification and crystallization of HNE have been published protease inhibitor replacement is one potential therapeutic (11). Crystals grew as large hexagonal prisms diffracting at approach currently under investigation (5). We have focused 1.84-A resolution in space group P63, with unit cell dimen- our attention on small molecular weight inhibitors ofHNE to sions, a = b = 74.53 A, c = 70.88 A, a = 83 = 90", y = 120°. The supplement the action of a, protease inhibitor and to retard diffractometer data collection and data reduction protocol the progression of the disease. Such inhibitors might also used have been described (17). allow a clarification ofthe role ofHNE in other diseases, such To solve the structure, over 30 different heavy-atom as the adult respiratory distress syndrome (6) and rheumatoid derivative experiments, both soaks and cocrystallizations, arthritis (7). were tried. In all cases, visual inspection of precession HNE is a basic (pI > 10), single-chain glycoprotein of -25 photographs for intensity differences proved inconclusive, so kDa. It is a serine protease that prefers small aliphatic amino all screening for heavy-atom derivatives had to be done by acids at the P1 11 position of substrates and inhibitors (9). The diffractometry. Four of these derivatives led to difference sequence of HNE has been determined, and it shows 43% Patterson maps, which were interpretable with the aid of a homology with that ofporcine pancreatic elastase (PPE) (10). vector verification procedure (18). Table 1 summarizes these Here we present the three-dimensional structure at pH 7 of heavy-atom substitution, refinement, and multiple isomor- the complex of HNE inactivated by a peptidyl inhibitor, phous replacement results. Electron density maps based on methoxysuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone this phase information proved uninterpretable, however. To (MSACK). [Crystals ofnative HNE have not proved suitable supplement the missing connectivity in the maps, a system- for high-resolution diffraction studies (11).] Coordinates for the HNE-MSACK complex have been deposited in the Abbreviations: HNE, human neutrophil elastase; PPE, porcine Brookhaven Protein Data Bank (12) for unrestricted distri- pancreatic elastase; MSACK, methoxysuccinyl-Ala-Ala-Pro-Ala bution. Bode et al. (13) have reported a structure of HNE in chloromethyl ketone; L-647,957, 3-acetoxymethyl-7a-chloro- complex with the third domain of turkey ovomucoid, a 3-cephem4carboxylate-1,1-dioxide tert-butyl ester (CAS registry macromolecular inhibitor, at pH 10. Our results appear number 95672-01-8). qualitatively in agreement with their published description of tTo whom reprint requests should be addressed at: Merck Sharp & Dohme Research Laboratories, P.O. Box 2000 (RY80M-203), Rah- way, NJ 07065. The publication costs of this article were defrayed in part by page charge 11P1..*P, refer to amino acid residues of the inhibitor, moving away payment. This article must therefore be hereby marked "advertisement" from the scissile bond in the N-terminal direction. Corresponding in accordance with 18 U.S.C. §1734 solely to indicate this fact. binding sites on the enzyme are designated S1. .S,, (8). 7 Downloaded by guest on September 25, 2021 8 Biochemistry: Navia et al. Proc. Natl. Acad. Sci. USA 86 (1989) Table 1. Heavy-atom refinement parameters for the structure of HNE in complex with MSACK* Derivativet Site x y z Occupancyt B, A2 HFMA (Hg) 1 0.114 0.778 0.000 60.2 50.2 HMNP (Hg) 1 0.994 0.632 0.115 38.4 13.1 2 0.038 0.644 0.041 51.7 26.7 HCMI (I) 1 0.442 0.254 0.748 53.0 10.6 H13S (I) 1 0.199 0.442 0.273 94.5 10.9 2 0.148 0.543 0.161 77.8 9.0 3 0.226 0.514 0.293 75.4 12.0 Figure of merit of resolution d, A 12.4 9.0 7.1 5.8 4.9 4.3 3.8 No. of reflections 47 105 169 255 356 509 600 Mean figure of merit 0.80 0.75 0.77 0.74 0.74 0.62 0.51 *Explanation of the parameters can be found in ref. 19. tHFMA: MSACK-inactivated HNE was crystallized as described (11), in the presence of 2 mM phenylmercury (II) acetate. Setting z = 0.0 for HFMA defines the origin. HMNP: Native, preformed HNE-MSACK crystals were exchanged into 2.0 M sodium/potas- sium phosphate at pH 7.0 and were treated anaerobically for 3 days with carbon disulfide as described by Mowbray and Petsko (20). The colorless CS2-treated crystals were transferred into 0.6 ml of a 10 mM (saturated) solution of 2-chloromercuri-4-nitrophenol (K & K) FIG. 1. Difference electron density map of the active site region in 2.0 M sodium/potassium phosphate at pH 7.0. Crystals turned of the complex of HNE inactivated by the inhibitor MSACK. The bright yellow on standing overnight and remained that way after map was computed with coefficients (1Fol - IFcI) and phases 40, extensive back-soaking, indicating covalent heavy-atom binding. where the inhibitor and active site residues His-57 and Ser-195 were Two mercury sites were ultimately and unexpectedly found in excluded from the calculation to minimize bias. These excluded association with residues His-25 and His-71. Note that HNE has no coordinates are plotted in white and are superimposed on the lysines (10) and a blocked N terminus. HCMI: HNE was inactivated difference map, which is drawn at 3.0o, in blue and 2.0oa in orange. with the inhibitor p-iodoanilidosuccinyl-Ala-Ala-Pro-Ala chloro- methyl ketone, an iodinated analog of MSACK specifically synthe- HNE observed structure factor data by using the program sized as a single-site, rational, heavy-atom derivative. Crystals were CORELS (25). The sequence of HNE (10) was then imposed on grown as described (11). HU3S: Native preformed HNE-MSACK the transformed PPE model coordinates, which were subse- crystals were exchanged into 2.0 M sodium/potassium phosphate at quently refitted onto the multiple isomorphous replacement pH 7.0. These were soaked overnight in the dark at room temper- ature in the exchange solution, which was made 4 mM in 12 and 10 map by using the graphics modeling program FRODO (26). A mM in KI, essentially as described (21). restrained least-squares refinement was applied to these tGiven in terms of electrons, when HCMI (see footnote t) is given preliminary HNE coordinates in shells of increasing resolu- the full occupancy (53 e-) of a bound iodine atom.
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