Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7-11, January 1989 Biochemistry Structure of human neutrophil elastase in complex with a 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 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 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 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. tion by using the PROLSQ programs of Hendrickson and Konnert (24) under the control of an in-house expert system atic six-dimensional real-space rotation and translation program to be described elsewhere. A model of the reacted search (22, 23) was carried out by using the coordinates ofthe MSACK inhibitor was initially fit to difference electron related structure of PPE. PPE had been refined at 1.84-A density and was refined as a second polypeptide chain vs. resolution by a restrained least-squares procedure (24) to an appropriately modified N- and C-terminal dictionary entries. R factor of 16.5% (J.P.S. and M.A.N., unpublished results). The positions of 217 bound water molecules were obtained A unique match was obtained at 16 standard deviations above from difference electron density maps (subject to reasonable the average background correlation levels, a result that hydrogen-bonding geometry) and were refined with variable compares quite favorably with previous experience (22, 23). occupancy. Ultimately, refinement led to an R factor of The location and orientation of these transformed PPE 16.4% at 1.84-A resolution while preserving idealized geom- coordinates were adjusted by rigid body refinement vs. the etry. These results are summarized in Table 2. Table 2. Preliminary restrained least-squares refinement data for the HNE complex with the inhibitor MSACK Root-mean-square deviation from ideality R* factor, Bond Angle Resolution Reflections, Atoms, Bound Refinement % distance, A distance, A range, A no. no. waters, no. Target at 0.020 0.030 Initial model 43.2 0.016 0.036 10-2.5 6,887 1841 CORELS 37.6 10-2.8 11,369 1841 - PROLSQ Start 36.0 0.014 0.036 10-2.2 11,856 1841 End 16.4 0.019 0.038 8-1.84 16,828 1920 217 *The R factor is defined by the expression R = I || Fobsl - IFcacll/ Fcaicl, where Fobs are the observed structure factors and Fcaic are the corresponding calculated structure factors. tCovalently bonded heteroatoms must be so designated in PROLSQ and assigned target bond distances. These were 1.43 A from Ser-195 OG to AlaP, C, and 1.47 A from His-57 NE2 to AlaPj CT (derived from a survey of relevant small molecule structures in the Cambridge Crystallographic Data File). To minimize bias, very loose restraints were imposed on these bonds, corresponding to an allowed standard deviation of 0.5 A (and separately, 0.1 A with equivalent results). Downloaded by guest on September 25, 2021 Biochemistry: Navia et al. Proc. NatL. Acad. Sci. USA 86 (1989) 9 RESULTS AND DISCUSSION may be the result of partial regeneration of enzyme activity Structure of the Enzyme-Inhibitor Complex. Structures of over the 3-4 weeks that passed between the formation ofthe serine protease complexes inactivated by peptide chloro- HNE-MSACK complex, its crystallization, and the comple- methyl ketones have been reported (27-30) but none at the tion of data collection. These bonds refine to reasonable level ofresolution and refinement reached here. A difference distances of 1.6 A and 1.5 A, respectively. electron density map ofthe HNE-MSACK complex is shown Fig. 2 shows the P1 to P3 residues ofthe inhibitor (in green) in Fig. 1, with refined coordinates superimposed. Table 3 lists bound to HNE (in red) at corresponding S, to S3 sites that a number ofkey distances and angles in the active site region have been defined by the structures ofinhibitor complexes in and contains a schematic diagram that defines the nomen- other serine proteases (for example, see ref. 32). Bond clature used. The chymotrypsinogen sequence numbering distances for this region are shown in Table 3. Electron convention (31) is used throughout. density beyond P4 in the HNE complex is considerably The observed structure is consistent with the currently weaker than that ofthe rest ofthe inhibitor. Still, coordinates accepted mechanism of action for the inhibition of serine fit to this density refine well (although with higher tempera- proteases by chloromethyl ketones (9). Clear bridging density ture factors) and trace out a substrate-like conformation to corresponds to a covalent bond between Ser-195 OG and the the surface of the enzyme. This interpretation is indepen- ketal C carbon of the AlaP, residue of the inhibitor. The dently supported by the position of the terminal iodine in the geometry around the ketal carbon is tetrahedral (see Table 3). HNE complex with the heavy-atom derivative analog, p- We also observe somewhat weaker density between His-57 iodoanilidosuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone. NE2 and the terminal CT carbon of the AlaP1 residue. This Given the covalent links shown in Fig. 1 between the inhibitor and the catalytic residues Ser-195 and His-57 of Table 3. Selected distances and angles for the complex of human HNE, one can infer that the MSACK binding configuration neutrophil elastase with the inhibitor MSACK observed is close to the productive one and has been Bond angles in degrees preserved in the course of the inhibition reaction. Earlier Ser-195 OG-AlaPj C-AlaP, 0 97 crystallographic studies with inhibitor and cleavage product Ser-195 OG-AlaP, C-AlaP1 CA 105 complexes in PPE (33-36) showed a variety of unusual Ser-195 OG-AlaP, C-AlaP, CT 115 binding configurations-presumably a reflection of the shal- AlaP, CA-AlaPj C-AlaP, CT 117 low depth of the S, specificity pocket-and suggested that AlaP, O-AlaP, C-AlaP, CT 108 elastase might be atypical among the serine proteases. Our AlaP, O-AlaPj C-AlaP, CA 112 results strongly support the view that the elastases are Ser-195 CB-Ser-195 OG-AlaPj C 119 "conventional" -like serine proteases in their His-57 NE2-AlaP, CT-AlaP, C 115 mode of productive binding as well as in the configuration of AlaP, C-AlaP, CA-AlaPj CB 112 their catalytic groups. AlaP, C-AlaP, CA-AlaP, N 111 Bond distances in angstroms His-57 ND1-Asp-102 OD2 2.7 (H-bond, enzyme) Ser-214 OG-Asp-102 OD2 2.8 (H-bond, enzyme) His-57 N-Asp-102 OD1 2.9 (H-bond, enzyme) Ala-56 N-Asp-102 ND1 2.9 (H-bond, enzyme) AlaP, C-AlaP, 0 1.4 (covalent bond, inhibitor) AlaP, C-AlaP, CT 1.6 (covalent bond, inhibitor) AlaP, C-AlaP, CA 1.5 (covalent bond, inhibitor) AlaP, C-Ser-195 OG 1.5 (covalent bond to protein) AlaP, CT-His-57 NE2 1.6 (covalent bond to protein) AlaP, O-Gly-193 N 3.1 (H-bond, oxyanion hole) AlaP O-Ser-195 N 3.1 (H-bond, oxyanion hole) AlaP, O-Wat-108 0 2.5 (H-bond, water molecule) AlaP, N-Ser-214 0 2.9 (H-bond, f8 sheet) AlaP, CB-Cys-191 0 3.8 (inhibitor-enzyme contact) AlaPj CB-Ser-214 0 3.9 (inhibitor-enzyme contact) AlaP3 O-Val-216 N 3.1 (H-bond, ,B sheet) AlaP3 N-Val-216 0 2.9 (H-bond, 8 sheet) AlaP4 O-Wat-108 0 2.5 (H-bond, water molecule) AlaP4 O-Wat-502 0 2.7 (H-bond, water molecule) AlaP4 CA-Arg-217A CG 3.8 (inhibitor-enzyme contact) AlaP4 O1-Arg-217A CA 3.8 (inhibitor-enzyme contact) FIG. 2. Coordinates of the MSACK inhibitor (in green) and the AlaP4 O1-Wat-019 0 3.2 (H-bond, water molecule) active site and specificity regions of HNE (in red), shown superim- MeoSuc OT1-Wat-383 0 3.1 (H-bond, water molecule) posed on the equivalent coordinates ofPPE (in blue). PPE coordinates were refined at 1.84-A resolution to an R factor of 16.5% (J.P.S. and MeoSuc CT-Tyr-224 OH 2.9 (short contact) M.A.N., unpublished results). The RIGID transformation option within The schematic diagram of the immediate active site region, which the graphics program FRODO (26) was used to superimpose the defines the nomenclature used, is shown below. a-carbon atoms of critical residues His-57, Asp-102, Phe/Gln-192, Gly-193, Asp-194, Ser-195, Ala/Thr-213, Ser-214, Phe-215, Val-216, N- and Asp/Thr-226 in HNE and PPE, respectively, and ofCys residues CB~ s 42, 58, 136, 168, 182, 191, 201, and 220. Average root-mean-square AIaP CA CD1 His57 I Il N44 deviation in the position of the 19 target atoms was 0.48 A. The O-C-CT- NE2 CG-CB deletion of PPE residue Ser-217 in HNE induces a large readjustment in the structure of that entire loop and in the position and direction of CA G CE2-N( CA the chain of residue Arg-217A. Residues Val-216 and Val-190 block CB Ser195 access to Asp-226 from the catalytic site at the mouth ofthe S, pocket. Downloaded by guest on September 25, 2021 10 Biochemistry: Navia et al. Proc. Nati. Acad. Sci. USA 86 (1989) Overlapping the structures of HNE (red) and PPE (blue) in vicinity ofS1 and within the specificity pocket itself, shifts the Fig. 2 shows the two to be quite similar, particularly relative position ofthe mouth ofthe pocket and makes it more in the immediate active site region. The largest differences hydrophobic. The inhibitor L-647,957 readily accommodates between the structures are observed in the S4 and S5 binding these changes (particularly in the acylated open f3-lactam ring regions. The deletion of PPE residue Ser-217 in HNE form). This assertion is supported by the similar inactivation significantly alters the overall conformation of the corre- behavior of L-647,957 with both PPE and HNE. L-647,957 sponding loop as well as the position and direction of inactivates PPE and HNE by an acylation mechanism (k2 = Arg-217A, such that the path traced by MSACK in the HNE 0.02 sec' and 0.01 sec'; K1 = 0.3 jLM and 0.6 AM, complex would now interpenetrate the PPE structure. respectively), yielding a stable, inactive enzyme in both cases Replacement of the PPE residue Thr-226 by Asp in the Si with a very small regeneration rate (kr < 10-4 min-) at 250C specificity pocket of HNE (10) suggested the design of (38, 39). superior enzyme inhibitors incorporating synthetic positively Residue Gln-192 in PPE is replaced by Phe in HNE. Even charged P1 side chains. These would be analogous to the though the residues are roughly isosteric, the direct hydrogen inhibitors, which use the naturally occurring amino bond observed in PPE between Gln-192 and the sulfone acids Lys and Arg to interact with residue Asp-189 at the oxygen ofL-647,957 cannot be duplicated. Instead, a sulfone bottom of the trypsin S1 pocket. On examination of the oxygen ofthe inhibitor interacts with the edge ofthe Phe-192 structure of HNE, however, we found the path through the ring in HNE in an orientation similar to that described for Si pocket to Asp-226 to be tortuous at best, blocked in part carbonyls and phenyl groups by Thomas et al. (40). Their by residues Val-190 and Val-216 (see Fig. 2). Asp-226 itself estimate of the binding energy for such an interaction tucks away from the active site region, deepening the SI pocket (as compared with PPE) in an area that is inaccessible to substrate and whose relevance to binding or catalysis is unclear. Asp-226 hydrogen bonds into an extensive network of the most highly ordered solvent atoms in the structure, which are hydrogen bonded in turn to the surrounding backbone atoms and to bulk solvent. The solvent network may serve to dissipate the negative charge of Asp-226, as shown for the binding of sulfate in the bacterial chemotactic protein (37). HNE Binding to f-Lactam Inhibitors. Fig. 3 shows the coordinates of the active site region of HNE, taken from the MSACK complex, superimposed on the complex ofPPE with the 18-lactam inhibitor L-647,957 (17). In the absence of a native structure for HNE, the similarity between the two enzymes suggests that the binding of MSACK did not significantly alter the structure of HNE. The replacement of Gly-190 in PPE by Val in HNE and of Thr-213 by Ala, in the

FIG. 4. Distribution of positively charged Arg and Lys residues (in blue) on HNE and rat mast cell protease II (46), the two granule-associated enzymes of known three-dimensional structure. FIG. 3. Overlap ofthe HNE coordinates from the HNE-MSACK Both enzymes are shown in equivalent orientations. The catalytic complex with coordinates of the PPE complex with the .8-lactam triad residues His-57, Asp-102, and Ser-195 are shown in green and inhibitor L-647,957 (17). Colors are as in Fig. 2, except that green identify the two active site regions. (Upper) HNE a-carbon atoms are now represents the -3-lactam inhibitor. The same 19 target atoms shown in red. Positively charged Arg residues [HNE has no Lys (10)] listed above were used in this superimposition, with a root- are located exclusively on the periphery of the molecule and form a mean-square deviation of0.49 A. In the immediate area ofthe active roughly three-sided belt around it. (Lower) Rat mast cell protease II site where most of the (-lactam inhibitor interactions take place, a-carbon atoms are shown in violet. Here the positively charged HNE and PPE are quite similar (white areas indicate areas where red residues form a somewhat less-distinct belt, with a substantial degree and blue colors mix). of clumping towards the bottom of the molecule in this view. Downloaded by guest on September 25, 2021 Biochemistry: Navia et al. Proc. NatL. Acad. Sci. USA 86 (1989) 11 represents a substantial portion of that expected from a 11. Williams, H. R., Lin, T.-Y., Navia, M. A., Springer, J. P., hydrogen bond. McKeever, B. M., Hoogsteen, K. & Dorn, C. P., Jr. (1987) J. Packaging of EINE in Neutrophil Storage Granules. The Biol. Chem. 262, 17178-17181. cationic secretory enzymes, such as HNE, the rat mast cell 12. Bernstein, F. C., Koetzel, T. F., Williams, G. J. G., Meyer, proteases I and II, and the family ofgranzymes, are normally E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shiman- ouchi, T. & Tasumi, M. (1977) J. Mol. Biol. 112, 535-542. found in electron-dense granule structures in, respectively, 13. Bode, W., Wei, A. Z., Huber, R., Meyer, E. F., Jr., Travis, J. the neutrophils (41), mast cells (42), and cytolytic T lympho- & Neumann, S. (1986) EMBO J. 5, 2453-2458. cytes (43). In neutrophils in particular, histochemical staining 14. Trainor, D. A. (1987) Trends Pharm. Sci. 8, 303-307. for sulfate is initially intense in immature granules, masked 15. Powers, J. C. (1983) Am. Rev. Respir. Dis. 127, S54-S58. upon maturation, and regained on degranulation (44). This is 16. Ranga, V., Kleinerman, J., Ip, M. P. C., Sorensen, J. & consistent with a model in which a matrix of negatively Powers, J. C. (1981) Am. Rev. Respir. Dis. 124, 613-618. charged proteoglycan acts as a scaffold for the condensation 17. Navia, M. A., Springer, J. P., Lin, T.-Y., Williams, H. R., of positively charged enzymes and as a means of controlling Firestone, R. A., Pisano, J. M., Doherty, J. B., Finke, P. E. & their auto-destructive potential (45). Since different popula- Hoogsteen, K. (1987) Nature (London) 327, 79-82. tions of enzymes can segregate to different populations of 18. Steigemann, W. (1974) Ph.D. Thesis (Technische Universitat, granules within the same cell type (41), however, some Munich). 19. Tulinsky, A., Park, C. H. & Rydel, T. J. (1985) J. Biol. Chem. specificity mechanism must still be postulated. 260, 10771-10778. With this in mind, we examined the surface distribution of 20. Mowbray, S. L. & Petsko, G. A. (1983) J. Biol. Chem. 258, the positively charged amino acids Arg and Lys in HNE and 5634-5637. rat mast cell protease 11 (46), the only two cationic, granule- 21. Sigler, P. B. (1970) Biochemistry 9, 3609-3617. associated enzymes whose three-dimensional structures 22. Navia, M. A., Segal, D. M., Padlan, E. A., Davies, D. R., have been determined crystallographically. Fig. 4 shows the Rao, N., Rudikoff, S. & Potter, M. (1979) Proc. Nat!. Acad. a-carbon backbones of the two enzymes in similar orienta- Sci. USA 76, 4071-4074. tions with all Arg and Lys side chains displayed. In both 23. Suh, S. W., Bhat, T. N., Navia, M. A., Cohen, G. H., Rao, cases, charge distributions are highly asymmetric and might D. N., Rudikoff, S. & Davies, D. R. (1986) Proteins: Struct. Funct. Genet. 1, 74-80. well favor the binding of one vs. the other to a specific 24. Hendrickson, W. A. (1985) Methods Enzymol. 115, 252-270. complementary pattern of negative charges in the underlying 25. Sussman, J. L. (1985) Methods Enzymol. 115, 271-303. proteoglycan matrix of the storage granule. A cursory exam- 26. Jones, T. A. (1985) Methods Enzymol. 115, 157-171. ination of the aligned sequences of the cytolytic T-lymphocyte 27. Segal, D. M., Powers, J. C., Cohen, G. H., Davies, D. R. & granzymes (43) suggests that their association with granules is Wilcox, P. E. (1971) Biochemistry 10, 3728-3737. consistent with this model also. 28. Poulos, T. L., Alden, R. A., Freer, S. T., Birktoft, J. J. & Kraut, J. (1976) J. Biol. Chem. 251, 1097-1103. 29. James, M. N. G., Brayer, G. D., Delbaere, L. T. J. & Sielecki, CONCLUSION A. R. (1980) J. Mol. Biol. 139, 423-438. 30. Betzel, C., Pal, G. P., Struck, M., Jany, K. D. & Saenger, W. Peptide chloromethyl ketones are active and effective inhib- (1986) FEBS Lett. 197, 105-110. itors in animal models of emphysema (15) but present side 31. Hartley, B. S. (1970) Phil Trans. R. Soc. London Ser. B 257, effects that make them unsuitable for human therapeutic use. 77-86. The coordinates of the HNE-MSACK complex should in 32. James, M. N. G., Sielecki, A., Brayer, G. D., Delbaere, general facilitate efforts to design more benign peptidyl L. T. J. & Bauer, C. A. (1980) J. Mol. Biol. 144, 43-88. inhibitors for this target. Structural information on the 33. Shotton, D. M., White, N. J. & Watson, H. C. (1972) Cold behavior offl-lactam inhibitors of HNE has been inferred by Spring Harbor Symp. Quant. Biol. 36, 91-105. comparing HNE with the previously solved structure of a 34. Hassall, C. H., Johnson, W. H. & Roberts, N. A. (1979) /3-lactam complex with PPE. Finally, the distri- Bioorg. Chem. 8, 299-309. asymmetric 35. Hughes, D. L., Sieker, L. C., Bieth, J. & Dimicoli, J. L. (1982) bution of positively charged residues on the surface of HNE J. Mol. Biol. 162, 645-658. suggests a mechanism for the specific incorporation of the 36. Meyer, E. F., Jr., Radhakrishnan, R., Cole, G. M. & Presta, enzyme into primary granules in the neutrophil. L. G. (1986) J. Mol. Biol. 189, 533-539. 37. Pflugrath, J. W. & Quiocho, F. A. (1985) Nature (London) 314, We would like to thank J. B. Doherty and our colleagues in the 257-260. elastase inhibitor project for their continuing interest; B. L. Bush and 38. Lin, T.-Y., Williams, H. R., Navia, M. A., Springer, J. P., R. Blevins for graphics support; and J. C. Powers, G. M. Smith, and Hoogsteen, K., Shah, S. K., Finke, P. E., Doherty, J. B. & R. L. Stein for discussions and suggestions. We also thank Mrs. Firestone, R. A. (1987) Fed. Proc. Fed. Am. Soc. Exp. Biol. 46, Stacianne Fischbach for her assistance in preparing this manuscript. 2223. 39. Doherty, J. B., Ashe, B. M., Argenbright, L. W., Barker, 1. Janoff, A. (1985) Annu. Rev. Med. 36, 207-216. P. L., Bonney, R. J., Chandler, G. O., Dahlgren, M. E., Dorn, 2. Carrell, R. W. (1987) J. Clin. Invest. 78, 1427-1431. C. P., Jr., Finke, P. E., Firestone, R. A., Fletcher, D., Hag- 3. Garver, R. I., Mornex, J. F., Nukiwa, T., Brantly, M., Court- mann, W. K., Mumford, R., O'Grady, L., Maycock, A. L., ney, M., LeCocq, J. P. & Crystal, R. G. (1986) N. Engl. J. Pisano, J. M., Shah, S. K., Thompson, K. R. & Zimmerman, Med. 314, 762-766. M. (1986) Nature (London) 322, 192-194. 4. Janus, E. D., Phillips, N. T. & Carrell, R. W. (1985) Lancet i, 40. Thomas, K. A., Smith, G. M., Thomas, T. B. & Feldmann, 152-154. R. J. (1982) Proc. Nat!. Acad. Sci. USA 79, 4843-4847. 5. Weinbaum, G. & Damiano, V. V. (1987) Trends Pharm. Sci. 8, 41. Dami'ano, V. V., Kucich, U., Murer, E., Laudenslager, N. & 6-7. Weinbaum, G. (1988) Am. J. Pathol. 131, 235-245. 6. Hyers, T. M. & Fowler, A. A. (1986) Fed. Proc. Fed. Am. Soc. 42. Schwartz, L. B. & Austen, K. F. (1980) J. Invest. Dermatol. Exp. Biol. 45, 25-29. 74, 349-353. 7. Breedveld, F. C., Lafeber, G. J. M., Siegert, C. E. H., Vleem- 43. Jenne, D., Rey, C., Haefliger, J.-A., Qiao, B.-Y., Grosgurth, P. & ing, L.-J. & Cats, A. (1987) J. Rheumatol. 14, 1008-1012. Tschopp, J. (1988) Proc. Nat!. Acad. Sci. USA 85, 4814-4818. 8. Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. 44. Parmley, R. T., Doran, T., Boyd, R. L. & Gilbert, C. (1986) J. Commun. 27, 157-162. Histochem. Cytochem. 34, 1701-1707. 9. Powers, J. C. (1986) in Proteinase Inhibitors, eds. Barrett, 45. Avila, J. L. & Convit, J. (1976) Biochem. J. 160, 129-136. A. J. & Salvesen, G. (Elsevier, New York), pp. 55-152. 46. Reynolds, R. A., Remington, S. J., Weaver, L. H., Fischer, 10. Sinha, S., Watorek, W., Karr, S., Giles, J., Bode, W. & Travis, R. G., Anderson, W. F., Ammon, H. L. & Matthews, B. W. J. (1987) Proc. Nat!. Acad. Sci. USA 84, 2228-2232. (1985) Acta Crystallogr. B41, 139-147. Downloaded by guest on September 25, 2021