Proc. Nati. Acad. Sci. USAVol. 86, pp. 7-11, January 1989Biochemistry

Structure of human neutrophil elastase in complex with a peptidechloromethyl 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 ArthritisResearch, Rahway, NJ 07065

Communicated by David R. Davies, August 12, 1988 (received for review January 29, 1988)

ABSTRACT Human neutrophil elastase (HNE) has beenimplicated as a major contributor to tissue destruction invarious disease states, including emphysema. The structure ofHNE, at neutral pH, in complex with methoxysuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone (MSACK), has beensolved and refmed to anR factor of 16.4% at 1.84-A resolution.Results are consistent with the currently accepted mechanismof peptide chloromethyl ketone inhibition of serine proteases,in that MSACK cross-links the catalytic residues His-57 andSer-195. The structure of the HNE-MSACK complex is com-pared with that of porcine pancreatic elastase in complex withL-647,957, a fi-lactam inhibitor of both elastases. The distri-bution of positively charged residues on HNE is highiy asym-metric and may play a role in its specific association with theunderlying negatively charged proteoglycan matrix of theneutrophil granules in which the enzyme is stored.

Human neutrophil elastase (HNE) is capable of digesting theunderlying elastin structure of the alveolar walls of the lung(1). Normally, a, protease inhibitor, a naturally occurringprotein present in the lung (2) acts to suppress HNE-mediateddamage. However, if a, protease inhibitor is present inunusually low amounts or if it is nonfunctional, eitherbecause of an underlying genetic defect (3) or due to smoking(4), the development of emphysema can follow. Direct a,protease inhibitor replacement is one potential therapeuticapproach currently under investigation (5). We have focusedour attention on small molecular weight inhibitors ofHNE tosupplement the action of a, protease inhibitor and to retardthe progression of the disease. Such inhibitors might alsoallow a clarification ofthe role ofHNE in other diseases, suchas the adult respiratory distress syndrome (6) and rheumatoidarthritis (7).HNE is a basic (pI > 10), single-chain glycoprotein of -25

kDa. It is a serine protease that prefers small aliphatic aminoacids at the P1 11 position of substrates and inhibitors (9). Thesequence of HNE has been determined, and it shows 43%homology with that ofporcine pancreatic elastase (PPE) (10).Here we present the three-dimensional structure at pH 7 ofthe complex of HNE inactivated by a peptidyl inhibitor,methoxysuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone(MSACK). [Crystals of native HNE have not proved suitablefor high-resolution diffraction studies (11).] Coordinates forthe HNE-MSACK complex have been deposited in theBrookhaven Protein Data Bank (12) for unrestricted distri-bution. Bode et al. (13) have reported a structure ofHNE incomplex with the third domain of turkey ovomucoid, amacromolecular inhibitor, at pH 10. Our results appearqualitatively in agreement with their published description of

the molecule, although coordinates have not been availablefor a quantitative comparison.Due to the clinical importance ofHNE, numerous peptide-

based as well as nonpeptidyl inhibitors of this enzyme and ofthe related PPE have been examined (9, 14). Peptide chlo-romethyl ketones such as MSACK have been shown to beeffective in vivo in arresting the development of experimen-tally induced emphysema in animal models of the disease(15). They have also served as a standard of comparison fornewly developed inhibitors, even though they present seriousside effects (16). The structure of the inactivated HNE-MSACK complex, in defining peptide binding to the enzyme,should be useful in the development of safer inhibitors.Recently, we reported the three-dimensional structure ofL-647,957 (3-acetoxymethyl-7a-chloro-3-cephem-4-carboxy-late-1,1-dioxide tert-butyl ester), a f-lactam HNE inhibitor,in complex with PPE (17). By comparing the structures of thetwo complexes, we can extend with confidence the PPE,3-lactam inhibition results to HNE, the actual drug target ofinterest.

MATERIALS AND METHODS

HNE derived from human purulent sputum was purchasedfrom Elastin Products, Saint Louis. Details of the finalpurification and crystallization ofHNE have been published(11). Crystals grew as large hexagonal prisms diffracting at1.84-A resolution in space group P63, with unit cell dimen-sions, a = b = 74.53 A, c = 70.88 A, a = 83 = 90", y = 120°. Thediffractometer data collection and data reduction protocolused have been described (17).To solve the structure, over 30 different heavy-atom

derivative experiments, both soaks and cocrystallizations,were tried. In all cases, visual inspection of precessionphotographs for intensity differences proved inconclusive, soall screening for heavy-atom derivatives had to be done bydiffractometry. Four of these derivatives led to differencePatterson maps, which were interpretable with the aid of avector verification procedure (18). Table 1 summarizes theseheavy-atom substitution, refinement, and multiple isomor-phous replacement results. Electron density maps based onthis phase information proved uninterpretable, however. Tosupplement the missing connectivity in the maps, a system-

Abbreviations: HNE, human neutrophil elastase; PPE, porcinepancreatic elastase; MSACK, methoxysuccinyl-Ala-Ala-Pro-Alachloromethyl ketone; L-647,957, 3-acetoxymethyl-7a-chloro-3-cephem4carboxylate-1,1-dioxide tert-butyl ester (CAS registrynumber 95672-01-8).tTo whom reprint requests should be addressed at: Merck Sharp &Dohme Research Laboratories, P.O. Box 2000 (RY80M-203), Rah-way, NJ 07065.11P1..*P, refer to amino acid residues of the inhibitor, moving awayfrom the scissile bond in the N-terminal direction. Correspondingbinding sites on the enzyme are designated S1. .S,, (8).

7

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Proc. Natl. Acad. Sci. USA 86 (1989)

Table 1. Heavy-atom refinement parameters for the structure ofHNE in complex with MSACK*Derivativet Site x y z Occupancyt B, A2HFMA (Hg) 1 0.114 0.778 0.000 60.2 50.2HMNP (Hg) 1 0.994 0.632 0.115 38.4 13.1

2 0.038 0.644 0.041 51.7 26.7HCMI (I) 1 0.442 0.254 0.748 53.0 10.6H13S (I) 1 0.199 0.442 0.273 94.5 10.9

2 0.148 0.543 0.161 77.8 9.03 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 3.8No. of reflections 47 105 169 255 356 509 600Mean 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, preformedHNE-MSACK crystals were exchanged into 2.0 M sodium/potas-sium phosphate at pH 7.0 and were treated anaerobically for 3 dayswith carbon disulfide as described by Mowbray and Petsko (20). Thecolorless CS2-treated crystals were transferred into 0.6 ml of a 10mM (saturated) solution of 2-chloromercuri-4-nitrophenol (K & K)in 2.0 M sodium/potassium phosphate at pH 7.0. Crystals turnedbright yellow on standing overnight and remained that way afterextensive back-soaking, indicating covalent heavy-atom binding.Two mercury sites were ultimately and unexpectedly found inassociation with residues His-25 and His-71. Note that HNE has nolysines (10) and a blocked N terminus. HCMI: HNE was inactivatedwith the inhibitor p-iodoanilidosuccinyl-Ala-Ala-Pro-Ala chloro-methyl ketone, an iodinated analog of MSACK specifically synthe-sized as a single-site, rational, heavy-atom derivative. Crystals weregrown as described (11). HU3S: Native preformed HNE-MSACKcrystals were exchanged into 2.0M sodium/potassium phosphate atpH 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 10mM in KI, essentially as described (21).tGiven in terms of electrons, when HCMI (see footnote t) is giventhe full occupancy (53 e-) of a bound iodine atom.

atic six-dimensional real-space rotation and translationsearch (22, 23) was carried out by using the coordinates of therelated structure of PPE. PPE had been refined at 1.84-Aresolution by a restrained least-squares procedure (24) to anR factor of 16.5% (J.P.S. and M.A.N., unpublished results).A unique match was obtained at 16 standard deviations abovethe average background correlation levels, a result thatcompares quite favorably with previous experience (22, 23).The location and orientation of these transformed PPE

coordinates were adjusted by rigid body refinement vs. the

FIG. 1. Difference electron density map of the active site regionof the complex of HNE inactivated by the inhibitor MSACK. Themap was computed with coefficients (1Fol - IFcI) and phases 40,where the inhibitor and active site residues His-57 and Ser-195 wereexcluded from the calculation to minimize bias. These excludedcoordinates are plotted in white and are superimposed on thedifference map, which is drawn at 3.0o, in blue and 2.0oa in orange.

HNE observed structure factor data by using the programCORELS (25). The sequence ofHNE (10) was then imposed onthe transformed PPE model coordinates, which were subse-quently refitted onto the multiple isomorphous replacementmap by using the graphics modeling program FRODO (26). Arestrained least-squares refinement was applied to thesepreliminary HNE coordinates in shells of increasing resolu-tion by using the PROLSQ programs of Hendrickson andKonnert (24) under the control of an in-house expert systemprogram to be described elsewhere. A model of the reactedMSACK inhibitor was initially fit to difference electrondensity and was refined as a second polypeptide chain vs.appropriately modified N- and C-terminal dictionary entries.The positions of 217 bound water molecules were obtainedfrom difference electron density maps (subject to reasonablehydrogen-bonding geometry) and were refined with variableoccupancy. Ultimately, refinement led to an R factor of16.4% at 1.84-A resolution while preserving idealized geom-etry. These results are summarized in Table 2.

Table 2. Preliminary restrained least-squares refinement data for the HNE complex with the inhibitor MSACKRoot-mean-square deviation from ideality

R* factor, Bond Angle Resolution Reflections, Atoms, BoundRefinement % distance, A distance, A range, A no. no. waters, no.Target at 0.020 0.030Initial model 43.2 0.016 0.036 10-2.5 6,887 1841CORELS 37.6 10-2.8 11,369 1841 -PROLSQ

Start 36.0 0.014 0.036 10-2.2 11,856 1841End 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 Afrom Ser-195 OG to AlaP, C, and 1.47 A from His-57 NE2 to AlaPj CT (derived from a survey of relevant small moleculestructures in the Cambridge Crystallographic Data File). To minimize bias, very loose restraints were imposed on thesebonds, corresponding to an allowed standard deviation of 0.5 A (and separately, 0.1 A with equivalent results).

8 Biochemistry: Navia et al.

Proc. NatL. Acad. Sci. USA 86 (1989) 9

RESULTS AND DISCUSSIONStructure of the Enzyme-Inhibitor Complex. Structures of

serine protease complexes inactivated by peptide chloro-methyl ketones have been reported (27-30) but none at thelevel of resolution and refinement reached here. A differenceelectron density map ofthe HNE-MSACK complex is shownin Fig. 1, with refined coordinates superimposed. Table 3 listsa number of key distances and angles in the active site regionand contains a schematic diagram that defines the nomen-clature used. The chymotrypsinogen sequence numberingconvention (31) is used throughout.The observed structure is consistent with the currently

accepted mechanism of action for the inhibition of serineproteases by chloromethyl ketones (9). Clear bridging densitycorresponds to a covalent bond between Ser-195 OG and theketal C carbon of the AlaP, residue of the inhibitor. Thegeometry around the ketal carbon is tetrahedral (see Table 3).We also observe somewhat weaker density between His-57NE2 and the terminal CT carbon of the AlaP1 residue. This

Table 3. Selected distances and angles for the complex of humanneutrophil elastase with the inhibitor MSACK

Bond angles in degreesSer-195 OG-AlaPj C-AlaP, 0 97Ser-195 OG-AlaP, C-AlaP1 CA 105Ser-195 OG-AlaP, C-AlaP, CT 115AlaP, CA-AlaPj C-AlaP, CT 117AlaP, O-AlaP, C-AlaP, CT 108AlaP, O-AlaPj C-AlaP, CA 112Ser-195 CB-Ser-195 OG-AlaPj C 119His-57 NE2-AlaP, CT-AlaP, C 115AlaP, C-AlaP, CA-AlaPj CB 112AlaP, C-AlaP, CA-AlaP, N 111

Bond distances in angstroms

may be the result of partial regeneration of enzyme activityover the 3-4 weeks that passed between the formation of theHNE-MSACK complex, its crystallization, and the comple-tion of data collection. These bonds refine to reasonabledistances of 1.6 A and 1.5 A, respectively.

Fig. 2 shows the P1 to P3 residues of the inhibitor (in green)bound to HNE (in red) at corresponding S, to S3 sites thathave been defined by the structures of inhibitor complexes inother serine proteases (for example, see ref. 32). Bonddistances for this region are shown in Table 3. Electrondensity beyond P4 in the HNE complex is considerablyweaker than that of the rest of the inhibitor. Still, coordinatesfit to this density refine well (although with higher tempera-ture factors) and trace out a substrate-like conformation tothe surface of the enzyme. This interpretation is indepen-dently supported by the position of the terminal iodine in theHNE complex with the heavy-atom derivative analog, p-iodoanilidosuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone.Given the covalent links shown in Fig. 1 between the

inhibitor and the catalytic residues Ser-195 and His-57 ofHNE, one can infer that the MSACK binding configurationobserved is close to the productive one and has beenpreserved in the course of the inhibition reaction. Earliercrystallographic studies with inhibitor and cleavage productcomplexes in PPE (33-36) showed a variety of unusualbinding configurations-presumably a reflection of the shal-low depth of the S, specificity pocket-and suggested thatelastase might be atypical among the serine proteases. Ourresults strongly support the view that the elastases are"conventional" chymotrypsin-like serine proteases in theirmode of productive binding as well as in the configuration oftheir catalytic groups.

His-57 ND1-Asp-102 OD2Ser-214 OG-Asp-102 OD2His-57 N-Asp-102 OD1Ala-56 N-Asp-102 ND1AlaP, C-AlaP, 0AlaP, C-AlaP, CTAlaP, C-AlaP, CAAlaP, C-Ser-195 OGAlaP, CT-His-57 NE2AlaP, O-Gly-193 NAlaP O-Ser-195 NAlaP, O-Wat-108 0AlaP, N-Ser-214 0AlaP, CB-Cys-191 0AlaPj CB-Ser-214 0AlaP3 O-Val-216 NAlaP3 N-Val-216 0AlaP4 O-Wat-108 0AlaP4 O-Wat-502 0AlaP4 CA-Arg-217A CGAlaP4 O1-Arg-217A CAAlaP4 O1-Wat-019 0MeoSuc OT1-Wat-383 0MeoSuc CT-Tyr-224 OH

2.72.82.92.91.41.61.51.51.63.13.12.52.93.83.93.12.92.52.73.83.83.23.12.9

(H-bond, enzyme)(H-bond, enzyme)(H-bond, enzyme)(H-bond, enzyme)(covalent bond, inhibitor)(covalent bond, inhibitor)(covalent bond, inhibitor)(covalent bond to protein)(covalent bond to protein)(H-bond, oxyanion hole)(H-bond, oxyanion hole)(H-bond, water molecule)(H-bond, f8 sheet)(inhibitor-enzyme contact)(inhibitor-enzyme contact)(H-bond, ,B sheet)(H-bond, 8 sheet)(H-bond, water molecule)(H-bond, water molecule)(inhibitor-enzyme contact)(inhibitor-enzyme contact)(H-bond, water molecule)(H-bond, water molecule)(short contact)

The schematic diagram of the immediate active site region, which

defines the nomenclature used, is shown below.

N-CB~

AIaPs CA CD1 His57

I Il N44O-C-CT- NE2 CG-CB

CA G CE2-N( CA

CB Ser195

FIG. 2. Coordinates of the MSACK inhibitor (in green) and theactive site and specificity regions of HNE (in red), shown superim-posed on the equivalent coordinates ofPPE (in blue). PPE coordinateswere refined at 1.84-A resolution to an R factor of 16.5% (J.P.S. andM.A.N., unpublished results). The RIGID transformation option withinthe graphics program FRODO (26) was used to superimpose thea-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,and Asp/Thr-226 in HNE and PPE, respectively, and ofCys residues42, 58, 136, 168, 182, 191, 201, and 220. Average root-mean-squaredeviation in the position of the 19 target atoms was 0.48 A. Thedeletion ofPPE residue Ser-217 in HNE induces a large readjustmentin the structure of that entire loop and in the position and direction ofthe chain of residue Arg-217A. Residues Val-216 and Val-190 blockaccess to Asp-226 from the catalytic site at the mouth ofthe S, pocket.

Biochemistry: Navia et al.

Proc. Nati. Acad. Sci. USA 86 (1989)

Overlapping the structures ofHNE (red) and PPE (blue) inFig. 2 shows the two enzymes to be quite similar, particularlyin the immediate active site region. The largest differencesbetween the structures are observed in the S4 and S5 bindingregions. The deletion of PPE residue Ser-217 in HNEsignificantly alters the overall conformation of the corre-sponding loop as well as the position and direction ofArg-217A, such that the path traced by MSACK in the HNEcomplex would now interpenetrate the PPE structure.Replacement of the PPE residue Thr-226 by Asp in the Si

specificity pocket of HNE (10) suggested the design ofsuperior enzyme inhibitors incorporating synthetic positivelycharged P1 side chains. These would be analogous to thetrypsin inhibitors, which use the naturally occurring aminoacids Lys and Arg to interact with residue Asp-189 at thebottom of the trypsin S1 pocket. On examination of thestructure of HNE, however, we found the path through theSi pocket to Asp-226 to be tortuous at best, blocked in partby residues Val-190 and Val-216 (see Fig. 2). Asp-226 itselftucks away from the active site region, deepening the SIpocket (as compared with PPE) in an area that is inaccessibleto substrate and whose relevance to binding or catalysis isunclear. Asp-226 hydrogen bonds into an extensive networkof the most highly ordered solvent atoms in the structure,which are hydrogen bonded in turn to the surroundingbackbone atoms and to bulk solvent. The solvent networkmay serve to dissipate the negative charge of Asp-226, asshown for the binding of sulfate in the bacterial chemotacticprotein (37).HNE Binding to f-Lactam Inhibitors. Fig. 3 shows the

coordinates of the active site region of HNE, taken from theMSACK complex, superimposed on the complex ofPPE withthe 18-lactam inhibitor L-647,957 (17). In the absence of anative structure for HNE, the similarity between the twoenzymes suggests that the binding of MSACK did notsignificantly alter the structure ofHNE. The replacement ofGly-190 in PPE by Val in HNE and of Thr-213 by Ala, in the

FIG. 3. Overlap of the HNE coordinates from the HNE-MSACKcomplex with coordinates of the PPE complex with the .8-lactaminhibitor L-647,957 (17). Colors are as in Fig. 2, except that greennow represents the -3-lactam inhibitor. The same 19 target atomslisted above were used in this superimposition, with a root-mean-square deviation of 0.49 A. In the immediate area of the activesite where most of the (-lactam inhibitor interactions take place,HNE and PPE are quite similar (white areas indicate areas where redand blue colors mix).

vicinity of S1 and within the specificity pocket itself, shifts therelative position ofthe mouth ofthe pocket and makes it morehydrophobic. The inhibitor L-647,957 readily accommodatesthese changes (particularly in the acylated open f3-lactam ringform). This assertion is supported by the similar inactivationbehavior of L-647,957 with both PPE and HNE. L-647,957inactivates PPE and HNE by an acylation mechanism (k2 =0.02 sec' and 0.01 sec'; K1 = 0.3 jLM and 0.6 AM,respectively), yielding a stable, inactive enzyme in both caseswith a very small regeneration rate (kr < 10-4 min-) at 250C(38, 39).Residue Gln-192 in PPE is replaced by Phe in HNE. Even

though the residues are roughly isosteric, the direct hydrogenbond observed in PPE between Gln-192 and the sulfoneoxygen of L-647,957 cannot be duplicated. Instead, a sulfoneoxygen of the inhibitor interacts with the edge of the Phe-192ring in HNE in an orientation similar to that described forcarbonyls and phenyl groups by Thomas et al. (40). Theirestimate of the binding energy for such an interaction

FIG. 4. Distribution of positively charged Arg and Lys residues(in blue) on HNE and rat mast cell protease II (46), the twogranule-associated enzymes of known three-dimensional structure.Both enzymes are shown in equivalent orientations. The catalytictriad residues His-57, Asp-102, and Ser-195 are shown in green andidentify the two active site regions. (Upper) HNE a-carbon atoms areshown in red. Positively charged Arg residues [HNE has no Lys (10)]are located exclusively on the periphery of the molecule and form aroughly three-sided belt around it. (Lower) Rat mast cell protease IIa-carbon atoms are shown in violet. Here the positively chargedresidues form a somewhat less-distinct belt, with a substantial degreeof clumping towards the bottom of the molecule in this view.

10 Biochemistry: Navia et al.

Proc. NatL. Acad. Sci. USA 86 (1989) 11

represents a substantial portion of that expected from ahydrogen bond.Packaging of EINE in Neutrophil Storage Granules. The

cationic secretory enzymes, such as HNE, the rat mast cellproteases I and II, and the family of granzymes, are normallyfound in electron-dense granule structures in, respectively,the neutrophils (41), mast cells (42), and cytolytic T lympho-cytes (43). In neutrophils in particular, histochemical stainingfor sulfate is initially intense in immature granules, maskedupon maturation, and regained on degranulation (44). This isconsistent with a model in which a matrix of negativelycharged proteoglycan acts as a scaffold for the condensationof positively charged enzymes and as a means of controllingtheir auto-destructive potential (45). Since different popula-tions of enzymes can segregate to different populations ofgranules within the same cell type (41), however, somespecificity mechanism must still be postulated.With this in mind, we examined the surface distribution of

the positively charged amino acids Arg and Lys in HNE andrat mast cell protease 11 (46), the only two cationic, granule-associated enzymes whose three-dimensional structureshave been determined crystallographically. Fig. 4 shows thea-carbon backbones of the two enzymes in similar orienta-tions with all Arg and Lys side chains displayed. In bothcases, charge distributions are highly asymmetric and mightwell favor the binding of one vs. the other to a specificcomplementary pattern of negative charges in the underlyingproteoglycan matrix of the storage granule. A cursory exam-ination of the aligned sequences of the cytolytic T-lymphocytegranzymes (43) suggests that their association with granules isconsistent with this model also.

CONCLUSIONPeptide chloromethyl ketones are active and effective inhib-itors in animal models of emphysema (15) but present sideeffects that make them unsuitable for human therapeutic use.The coordinates of the HNE-MSACK complex should ingeneral facilitate efforts to design more benign peptidylinhibitors for this target. Structural information on thebehavior of fl-lactam inhibitors ofHNE has been inferred bycomparing HNE with the previously solved structure of a/3-lactam complex with PPE. Finally, the asymmetric distri-bution of positively charged residues on the surface of HNEsuggests a mechanism for the specific incorporation of theenzyme into primary granules in the neutrophil.

We would like to thank J. B. Doherty and our colleagues in theelastase inhibitor project for their continuing interest; B. L. Bush andR. Blevins for graphics support; and J. C. Powers, G. M. Smith, andR. L. Stein for discussions and suggestions. We also thank Mrs.Stacianne Fischbach for her assistance in preparing this manuscript.

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