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Crystal structure of human macrophage elastase (MMP-12) in complex with a hydroxamic acid inhibitor 1
doi:10.1006/jmbi.2001.4953 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 312, 743±751
Crystal Structure of Human Macrophage Elastase (MMP-12) in Complex with a Hydroxamic Acid Inhibitor Herbert Nar1*, Karlheinz Werle2, Margit M. T. Bauer1, Horst Dollinger1 and Birgit Jung3 1
Department of Medicinal Chemistry, Boehringer Ingelheim Pharma KG Biberach, Germany 2
SibTeq Stable Isotope Biotechnique GmbH, Munich Germany 3 Department of Pulmonary Disease, Boehringer Ingelheim Pharma KG, Ingelheim Germany
Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in in¯ammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S10 -speci®city pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open speci®city pockets. The two crystallographically independent molecules adopt different conformations of the S10 -loop and its neighbouring loop due to differing crystal packing environments, suggesting that ¯exibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with b-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or ®ll the upper part of the speci®city pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12. # 2001 Academic Press
*Corresponding author
Keywords: macrophage metalloelastase; matrix metalloproteinase; MMP-12; crystal structure; chronic obstructive pulmonary disease
Introduction Human macrophage metalloelastase (MMP-12) is a member of a subclass of the zinc proteinase family of the metzincins,1 ± 3 the matrix metalloproteinases (MMPs), which degrade components of the extracellular matrix (ECM) in processes like Abbreviations used: MMP-12, human macrophage elastase; MMP, matrix metalloproteinase; ECM, extracellular matrix. E-mail address of the corresponding author: [email protected] 0022-2836/01/040743±9 $35.00/0
embryonic development, reproduction, cellular migration and tissue remodelling.1 MMP-12 is active against a broad range of ECM components, including elastin, ®bronectin, collagen IV, laminin, entactin, chondroitin sulfate and heparan sulfate. MMP-12 degrades a1-antitrypsin, which is the major inhibitor of human neutrophil elastase, thereby enhancing the overall elastolytic activity. Further, MMP-12 contributes to the angiostatin generation.4,5 In adult tissue, macrophages are the major source of MMP-12. Several studies have demonstrated that MMP-12 is involved in in¯ammatory processes and contributes to tissue remodelling # 2001 Academic Press
744 and tissue destruction. In tissue samples of colitis ulcerosa, MMP-12 was found abundantly expressed in macrophages in the in¯amed area and in the vicinity of shedding mucosal epithelium, supporting a role of this enzyme for tissue invasion and epithelial cell shedding.6 MMP-12 also contributes to the remodelling of elastolytic areas. A prominent expression of MMP-12 was detected in macrophages invading the atherosclerotic lesions induced in cholesterol-fed rabbits.7 In abdominal aortic aneurysms, a high expression of MMP-12 was demonstrated in macrophages within the degenerating aortic media.8 Abundant expression of MMP-12 was found in the skin after ultraviolet photoprovocation in areas of elastolytic material.9 Further insights into the role of MMP-12 were provided by studies with MMP-12-de®cient mice. Macrophages from MMP-12-de®cient mice were unable to penetrate basement membranes.10 In addition, these animals were protected from developing lung emphysema after chronic cigarette smoke exposure, indicating that MMP-12 is required for extracellular matrix proteolysis and tissue invasion, and plays an important role in mice in lung tissue destruction.11 MMP-12 shares common structural domains with other MMPs. The most closely related human matrix metalloproteinases are stromelysin-1 (MMP3) and interstitial collagenase (MMP-1), each with 49 % identity with MMP-12. The molecular mass of the MMP-12 proenzyme is 54 kDa and comprises three domains. The N-terminal proenzyme domain I (9 kDa short signaling peptide) includes a highly conserved cysteine residue that coordinates the zinc in the proenzyme forms.12,13 Domain II is the catalytic domain (22 kDa) that contains the zinc-binding HExxHxxGxxH sequence motif.14,15 This domain is followed by the ``hemopexin-like'' C-terminal domain III (23 kDa), which exhibits sequence homology to vitronectin and hemopexin. The 54 kDa MMP-12 proenzyme is activated, like all other matrix metalloproteinases, with loss of the N-terminal prosequence, leaving an active 45 kDa enzyme. This active MMP-12 is then readily processed to a mature 22 kDa species with loss of the C-terminal hemopexin-like domain.16 The structures of the catalytic domain of several members of the MMP family have recently become available (for a review, see Bode et al.15). A structural comparison reveals that the S10 subsite is the best-de®ned pocket in these MMPs and consists of a hydrophobic pocket that varies greatly in its depth. MMP-1 and MMP-7, which have shallow S10 pockets, prefer small hydrophobic amino acids at the P10 position. In contrast, MMP-3 and MMP8, which have deep S10 pockets, can accommodate large and small P10 amino acids with similar ef®ciency.17 MMP-12 has a preference for amino acids with large aliphatic side-chains, particularly leucine in the P10 position. Aromatic or hydrophobic amino acids are preferred at the P1 site, with small hydro-
Crystal Structure of MMP-12
phobic residues (preferably alanine) occupying P3.18 Because of its obvious functions in lung pathobiology, MMP-12 is an interesting pharmaceutical target. Therefore, we initiated a research program to determine the 3D structure of MMP-12 to enable us to study its interaction with ligands for the design of potent and selective inhibitors.
Results and Discussion We report the X-ray crystal structure of MMP-12 in complex with a hydroxamic acid inhibitor. The protein structure consists of the catalytic domain of MMP-12 (residues 100-280 of the complete sequence of the macrophage metalloelastase precursor, EC 3.4.24.65, Swissprot P39900) with an N-terminal glycine residue as a cloning artefact. This sequence was expressed in Escherichia coli as inclusion bodies and refolded into its active state. We observed partial autolytic cleavage of MMP-12 during the refolding and puri®cation steps at residue L250 (data not shown). Using the protocol described in Materials and Methods, the puri®ed protein contained only minor amounts of the C-terminally truncated lower molecular mass contaminant. The protein crystals that were grown from this material consist of pure, catalytically active wild-type enzyme. MMP-12 crystallises in the orthorhombic space group I222 with two molecules in the asymmetric unit. The structure was solved by molecular replacement using a homology model that was based on the 3D structure of stromelysin-1 (MMP-3, PDB Ê resolution code 1hfs) and was re®ned to 2.6 A with good stereochemistry and crystallographic R-factors (Table 1). Apart from the 16 C-terminal residues (residue numbers 265-280), which are not visible and probably dynamically disordered, the molecule is completely de®ned in the electron density (Figure 1). The two independent molecules were kept very similar during re®nement, apart from residues 204214 and 240-249, which are in different crystalline environments and were re®ned independently. In the crystal lattice, the molecules pack with their catalytic clefts oriented to one another so that two symmetry equivalent inhibitor molecules are in close contact. Molecular structure of MMP-12 As expected, MMP-12 adopts a fold that is highly similar to the other Zn-proteinases of the MMP protein family consisting of a ®ve-stranded b-sheet and three a-helices (Figure 2). The catalytic zinc ion is ligated by three histidine residues from the sequence motif HExxHxxGxxH at the C-terminal end of the central helix hB and the turn following it. b-Strand sIV functions as the template with which substrate peptide are associated in a functional complex via main-chain hydrogen bonding. In addition to the catalytic zinc ion, there is a struc-
Crystal Structure of MMP-12
745
Figure 1. Stereo representation of the active site with the zinc ligation sphere, the template strand residues and bound inhibitor superimposed on the experimental 2Fo ÿ Fc omit map contoured at 1.2s.
tural zinc ion and three calcium ions bound by the polypeptide chain. The loop following the Met-turn structure in the MMPs1 circumscribes the S10 -subsite that is the most prominent feature of the catalytic site because it forms a deep pocket and is the main speci®city determinant of the MMPs. The S10 -loop structure in MMP-12 (residues 240-250) is well ordered in both crystallographically independent molecules in the crystal lattice. Residues D244 to F248 at the Cterminal end of the S10 -loop form an a-helical turn structure similar to the analogous region in MMP8. It is stabilized mainly by hydrophobic interactions of residues V243, I245 and F248 with F213 and L214 of the neighbouring loop and a hydrogen bond formed by the strictly conserved Y240 OH with the backbone NH moiety of N211 (Figure 3). A different crystal packing environment for the two independent molecules induces a slightly different position of the C-terminal end of the loop Ê shift and a dis(residues 243-249) comprising a 1 A torsion of the helical turn in molecule A. B-factors indicate a higher than average mobility of the Cterminal part of the S10 loop in molecule B, which faces a solvent channel in the lattice, while this is not the case for molecule A, probably because here crystal contacts to a symmetry-related molecule stabilize a more rigid structure of the loop. Structural rearrangements upon ligand binding19 as well as S10 loop mobility are detected in X-ray and dynamic NMR-studies of MMP-13.20 The above observations of ¯exibility or adjustment of this part of the S10 -loop are consistently seen in other MMPstructures and are therefore probably a common feature for all MMPs. The sequence stretch of residues 204-214 between secondary structural elements sV and hB is located next to the S10 -loop and forms hydrophobic and hydrogen bond interactions with it. Its lower end exhibits stuctural differences in molecules A and B about residue H206, again
induced by different crystal packing environments. The key residue for the structuring of this loop, T204, forms hydrogen bonds to the carbonyl groups of H206 and G208 in molecule B, but only to H206 CO in molecule A, because here the mainchain conformation of residue G208 directs its carbonyl group into bulk solvent. The side-chain of H206 is in two conformations, differing in the w1-torsion angle, which is energetically more favourable in molecule A, whereas in molecule B the unfavourable conformation is compensated by the concomitant formation of a hydrogen bond of neighbouring T205 CO to K148 Nx of a symmetryrelated molecule. The N terminus of the expressed construct of the MMP-12 catalytic domain, residues F100 REMP104, preceded by a glycine residue that originates from the used expression vector, is a wellordered structure that is main-chain hydrogen bonded to a sequence stretch around the zinc-ligating H228 and forms a short, two-stranded antiparallel b-sheet. F100 NH is bound to the carboxylate group of D253, the ®rst of a conserved tandem of aspartate residues positioned in the ®rst turn of helix hC. This interaction is reminiscent of the interaction of the charged N terminus of a ``superactivated'' form of neutrophil collagenase (MMP8).21 The N-terminal glycine residue is within H-bonding distance from S230 OH. The ordered structure of the MMP-12 N terminus is in fact identical with the N termini of the F79-form of MMP-8 (PDB code 1kbc) and of matrilysin (PDB code 1mmq). It may represent the structure of the mature enzyme and may be more active than any N-terminally truncated forms. Residue M103, which is unique to MMP-12, is thereby positioned next to H222 and forms, together with residues P104 GGP107 and G186 on strand sIV, a major part of the unprimed side of the active-site cleft.
746
Crystal Structure of MMP-12
respectively), for MMPs 3, 8 and 13 this residue is leucine and the pocket is much larger. In MMP-12, this respective residue is leucine (L214). The conformation of the MMP-12 S10 -loop is very similar to the loop of MMP-8 especially because both proteins exhibit one helical turn at residues D244-F248 (MMP-12 sequence numbers) at the bottom end of the S10 -subsite. However, the MMP-8 S10 -subsite is closed at the far side due to the presence of an arginine residue (R222, V243 in MMP-12), while in MMP-12 the S10 -pocket extends into a channel that reaches through the protein to the far side of the protein surface. Thus, with respect to the 3D shape of the S10 -pocket, MMP-12 more closely resembles MMPs-3 and 13 in having a large open speci®city pocket. Inhibitor-protein interactions
Figure 2. Molecular structure of MMP-12 in complex with CGS27023A. (a) Ribbon representation of the catalytic domain structure with the bound metal ions, which are coloured blue (zinc) and grey (calcium). (b) Surface representation.
Comparison to other MMPs A comparison of the structurally characterized members of the MMP family reveals a topologically homogeneous ensemble of 3D structures with almost identical positions of secondary structural elements and loops (Figure 4). The largest deviations occur in the S10 -loop segment and the neighbouring loop, regions that are sequentially most divergent. The size of the S10 -pocket is different in the various MMPs because of the sequence variability and length of the S10 -loop and the type of amino acid positioned four residues N-terminal to the ®rst zinc-ligating histidine of the HExxHxxGxxH motif.19 While for MMP-1 and MMP-7 the pocket is small and closed due to the presence of a large residue at this position (arginine or tyrosine,
The hyroxamic acid inhibitor CGS27023A was originally discovered as an orally active stromelysin-1 (MMP-3) inhibitor that ef®ciently blocks cartilage degradation.22 Its af®nity toward mouse MMP-12 was recognized recently.23 This compound is a potent inhibitor of various members of the MMP family exhibiting only weak selectivity against MMPs 1, 2, 7, 8, 9, 12 and 13 (IC50 values 49.5 nM, 9.1 nM, 16.9 nM, 106 nM, 4.4 nM, 4.3 nM, 2.0 nM, 4.3 nM respectively). 3D solution structures of MMPs 1, 3 and 13 with CGS27023A have been reported based on NMR data.20,24,25 In the present structure, CGS27023A is bound to the catalytic zinc ion, as expected in a bidentate fashion, by the two hydroxamate oxygen atoms, resulting in a trigonal bipyramidal coordination of the zinc ion (Figure 5). The remainder of the ligand occupies the primed side26 of the active-site cleft. The protonated hydroxamate oxygen forms an asymmetric bifurcated hydrogen bond with the side-chain carboxylate group of E219. The hydroxamate NH group is involved in a hydrogen bond with A182 and the sulfone group forms a hydrogen bond with one of its oxygen atoms to L181 NH, mimicking one of the canonical hydrogen bonds formed by peptide substrates of the enzyme with these b-strand IV residues. The other potential hydrogen bonding groups of the inhibitor face bulk solvent. The isopropyl substituent does not form any interaction with the protein and is mainly solventexposed, forming solely intramolecular contacts to the edge of the pyridyl ring. The pyridyl subtituent sticks out of the active-site cleft above P238 and forms a close contact of its o-CH group with the carbonyl group of the same residue, another example of a hydrogen bond formed by an aromatic ring system. The methoxyphenyl moiety is placed in the upper part of the S10 -pocket of the proteinase between residues L181, T215 and the zinc-ligating H218. The lower part of the pocket is left unoccupied by the inhibitor and is ®lled with an array of water molecules. As mentioned above,
Crystal Structure of MMP-12
747
Figure 3. Stereo representation of the structure of the catalytic site with bound ligand and the S10 -loop forming the speci®city pocket of the enzyme. CGS27023A reaches only halfway into the large cavity, which is otherwise ®lled by solvent.
the S10 -pocket is in fact a channel that connects the active-site cleft with the other side of the protein so that the observed ordered water molecules are in rapid exchange with bulk sovent through this channel. The bound conformation of the inhibitor is a low-energy conformation in which the torsion angles about the rotatable bonds are close to stereochemically ideal values. The observed binding mode of CGS27023A reveals that a favourable bound conformation, strong hydrogen bonding and hydrophobic interactions contribute to the strong af®nity for MMP12 (IC50 2 nM). On the other hand, all interactions described can be formed equally well with various
other members of the MMP-family, which in turn explains the missing or weak selectivity pro®le of CGS27023A. In the present structure, a major crystal contact is formed by the interaction of the active-site clefts of two symmetry-related molecules. The contacts made by the protein are exclusively polar, namely hydrogen bonds of H228 NdH to D175 OD, R101 NH1 to K177 CO and F171 CO to H172 NdH, all of which follow dyad symmetry and are therefore repeated twice for a given pair of MMP-12 dimers. Thereby, the pyridyl and isopropyl substituents of the two inhibitor molecules that occupy both active sites come into close contact. In fact, the pyridyl
Figure 4. Stereo representation of a superposition of the backbone ribbons of MMP-1 (PDB code 1HFC, magenta), MMP-2 (PDB code 1CK7, blue), MMP-3 (PDB code 1SLM, light blue), MMP-7 (PDB code 1MMQ, green), MMP-8 (PDB code 1JAP, yellow), MMP-13 (PDB code 966C, orange) with MMP-12 (red). The secondary structural elements superimpose almost perfectly. Structural diversity is most prominent in the S10 -loop region and the neighbouring loop (lower right) and at the N terminus (left).
748
Crystal Structure of MMP-12
large P10 -groups, which convey differences in inhibition of about one to three orders of magnitude for MMP-3, 8 and 13 (large open S10 -pockets) versus MMP-1 and 7 (smaller S10 -pockets). However, binding of inhibitors with large P10 -groups is not generally prohibitive, as shown for MMP-1, for which an induced ®t of the enzyme is found when it is complexed with such an inhibitor.19 This study shows that MMP-12 falls into the category of MMPs 8 and 13 with a large S10 -pocket. Any design of ligands with large P10 -subtituents should therefore yield selectivity towards the other group of MMPs. A distinction within the ®rst group, however, will have to be made using subtituents that bind to other subsites of the enzyme.
Materials and Methods Chemical synthesis of CGS27023A The synthesis of the MMP inhibitor CGS27023A was as described.22 Molecular cloning of the proteinase domain of MMP12
Figure 5. Enzyme-inhibitor interactions. (a) Close-up view of the active site and into the S10 -speci®city pocket of MMP-12, which reaches through the protein and opens up to the other side. (b) A representation of the interactions of CGS27023A with the enzyme.
rings are in a perfect aromatic stacking arrangeÊ . From ment with an inter-ring distance of 3.5 A this observation it may be inferred that the bound conformation of the ligand is perturbed by the described crystal contact. A comparison with the observed conformations of the inhibitor bound to the other MMPs in the NMR solution structures, however, shows very similar overall features. Based on the observed low-energy conformation of the inhibitor and the similarity to the solution structures of CGS27023A complexed to MMP-1, 3 and 13, we reason that the crystal packing forces do not perturb the bound inhibitor structure signi®cantly in the present structure. Implications for drug design and selectivity The potency of almost all reported MMP inhibitors relies strongly on a zinc-binding functionality, substrate-like hydrogen bonding along b-stand sIV and hydrophobic interactions made in the the S10 pocket. Most other subsites are shallow and exposed to solvent and thus not readily used for increasing af®nity.27 The design of selectivity of inhibitors of MMPs consequently is based mainly on the in¯uence of
The domain II (proteinase domain) of the human macrophage elastase 12 (amino acid residues 100-279) was ampli®ed by PCR with human-speci®c primers hMP1 (33mer): 50 -AAACCATGGGTTTCAGGGAAATGC CAGGGGGGC-30 and hMP2 (38mer): 50 -AAAGGATCCTATTAAGCTGGTTCTGAATTGTCAGGATT-30 from a human placental cDNA bank (Clonetech HL1075b) and cloned in the NcoI/BamHI restriction site of the multiple cloning site of the expression vector pET8C (Novagen) as described.28,29 The insert (567 bp) was fully sequenced for control and the plasmid pHME transformed to E. coli BL21 (DE3) pLysS (Novagen). Expression The cells were grown in a 7.5 l fermenter (BioFlo IV, New Brunswick Scienti®c) under online control of pH, O2 and temperature in slightly modi®ed commercial minimal medium M9 (Sigma, Deisenhofen). At an A600 of 1, the expression was induced by adding IPTG to 1 mM. At 12 hours after induction the cells were pelleted by centrifugation at 3000 g for 30 minutes and frozen (ÿ70 C). Purification The cells were resuspended in 1000 ml of lysis buffer (50 mM Tris-HCl, pH 7.4) and disrupted by sonication and incubation with lysozyme for one hour at 37 C. After three extensive washing steps with lysis buffer and lysis buffer containing 0.05 % (w/v) Brij 35 and 1 M urea, the inclusion bodies pellet was resuspended in 1000 ml of lysis buffer containing 8 M urea and incubated for one hour at 37 C. Insoluble material was removed by centrifugation at 50,000 g and the supernatant was dialyzed in four steps against 15 l of dialysis buffer (50 mM Tris, 30 mM NaCl, 10 mM CaCl2, 0.05 % Brij 35) with decreasing concentration of urea. Precipitated protein was removed by centrifugation at 8000 g for 30 minutes. The supernatant was loaded onto
749
Crystal Structure of MMP-12 Table 1. Data collection and re®nement statistics Space group I222 Ê) Unit cell dimensions (A a 67.4, b 87.2, c 169.2 Ê) Resolution limits (A 100.0-2.6 Total observations 57,864 Unique reflections 15,776 Completeness (%) Overall 100 Ê ) shell (2.60-2.55 A 100 Rmerge Overall 0.18 Ê shell 2.60-2.55 A 0.58 Protein atoms, water molecules, inhibitor atoms, metal ions 2606, 333, 54, 10 R factor (%) 19.6 25.7 Rfree Ê) Resolution range (A 20.0-2.6 Number of reflections (free set) 15,757 (797) Rms deviation from ideal geometry Ê) Bond length (A 0.006 Bond angles (deg.) 1.17 2 Ê ) Temperature factors (A B-value model Restrained individual B-factors All protein atoms 27.1 rms bonded B 0.8
a column containing the tripeptide hydroxamate H-ProLeu-Gly-NHOH (Bachem) coupled to CH-Sepharose 4B (Pharmacia) and the matrix was washed with 50 mM Tris (pH 7.0), 500 mM NaCl until no more protein could be detected in the ¯ow-through. The MMP12 was eluted with elution buffer (50 mM Tris (pH 11), 500 mM NaCl) into a vessel containing about one-tenth of the elution volume of 1 M Tris (pH 7.0). Crystallization The eluted protein was inhibited with CGS27023A, subsequently the protein buffer was exchanged with the help of PD10-columns (Pharmacia) against the crystallization buffer (20 mM Tris (pH 7.4), 200 mM NaCl, 2 mm CaCl2). After concentrating the protein sample to 4 mg/ ml, crystallization was carried out with the hanging drop vapour diffusion method in Q-plates (Hampton research). Hexagonal-shaped crystals up to a size of 0.2 mm 0.2 mm 0.1 mm were grown after four to ten days at 4 and 20 C with a reservoir containing 100 mM sodium-cacodylate (pH 6.5) and 1.4 M sodium acetate. Data collection Data were collected from cryo-cooled crystals which were cryo-protected by addition of 30 % (v/v) glycerol to the mother liquor. Two native data sets were collected from two different crystal specimens on our in-house rotating anode generator with Cu-Ka radiation and on the synchrotron wiggler Ê . The latter beamline 6B at DESY at wavelength 1.05 A Ê resolution was used for data set extending to 2.6 A re®nement of the structure. The crystal space group is I222 with cell dimensions of Ê , b 87.2 A Ê , c 169.2 A Ê with two crystallograa 67.4 A phically independent molecules in the asymmetric unit. Using a molecular mass of 21 kDa for the expressed version of the MMP-12 catalytic domain, the speci®c Ê 3/Da and the approxivolume (VM) calculated is 3.0 A
mate solvent content is 54 % (v/v).30 Data were integrated, scaled and merged with the HKL package.31 Molecular replacement The structure was solved using AMoRe32 with a homology model of MMP-12 derived from the crystal structure of stromelysin (PDB code 1HFS) which displays 49 % sequence homology to MMP-12. The two molecules could be placed in the asymmetric unit unambiguously. They are related to each other by an improper local symmetry with a rotation component of 50 about the crystallographic c-axis. Model building and refinement The ®rst electron density was computed with the homology model used for molecular replacement. Density averaging over the two independent molecules combined with solvent ¯ipping was performed using MAIN.33 The electron density at this stage was interpretable for most of the protein chain. Repeated cycles of model building, rephasing and averaging were performed until a complete structural model was obtained. Water molecules were added based on peaks in difference Fourier maps and geometric considerations. The model was re®ned using CNS34 using data Ê resolution. Restrained non-crysbetween 20.0 and 2.6 A tallographic symmetry was used throughout re®nement. In later stages, deviations between the two molecules were allowed in the sequence regions 204-214 and 240249, which have slightly different main chain positions and distinct side-chain conformations due to crystal packing effects. The re®ned MMP-12 model has an R-value of 19.6 % and an Rfree value of 25.7 %. The r.m.s. deviation of the Ê (0.11 A Ê for Ca atoms two copies for all atoms is 0.29 A leaving out the regions 204-214 and 240-249). Data collection, re®nement statistics and geometrical parameters for the ®nal model are presented in Table 1. Protein Data Bank accession code The coordinates and structure factors for the present MMP-12-CGS27023A complex were deposited in the Protein Data Bank, accession code 1JIZ.
Acknowledgements We thank Professor Walter StoÈcker, University of MuÈnster, Germany, for the af®nity puri®cation protocol and helpful discussions, Dr Thomas Fox for providing us with a homology model for MMP-12 and Rebecca Scherer and Andrea KuÈhn for technical support. We acknowledge the help of Hans Bartunik during data collection on the MPG/GBF wiggler beamline BW6/DORIS at DESY.
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Crystal Structure of MMP-12
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26. 27. 28.
29.
tural properties of matrix metalloproteinases. Cell. Mol. Life Sci. 55, 639-652. Shapiro, S. D., Kobayashi, D. K. & Ley, T. J. (1993). Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages. J. Biol. Chem. 268, 23824-23829. Shapiro, S. D. (1994). Elastolytic metalloproteinases produced by human mononuclear phagocytes. Potential roles in destructive lung disease. Am. J. Respir. Crit. Care Med. 150, S160-S164. Gronski, T. J., Jr, Martin, R. L., Kobayashi, D. K., Walsh, B. C., Holman, M. C. & Huber, M. et al. (1997). Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase. J. Biol. Chem. 272, 12189-12194. Lovejoy, B., Welch, A. R., Carr, S., Luong, C., Broka, C. & Hendricks, R. T., et al. (1999). Crystal structures of MMP-1 and -13 reveal the structural basis for slectivity of collagenase inhibitors. Nature Struct. Biol. 6, 217-221. Zhang, X., Gonnella, N. C., Koehn, J., Pathak, N., Ganu, V. & Melton, R. et al. (2000). Solution structure of the catalytic domain of human collagenase-3 (MMP-13) complexed to a potent non-peptidic sulfonamide inhibitor: binding comparison with stromelysin-1 and collagenase-1. J. Mol. Biol. 301, 513-524. Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S. & Piper, M. et al. (1994). Structural implications for the role of the N terminus in the ``superactivation'' of collagenases. A crystallographic study. FEBS Letters, 338, 227-233. MacPherson, L. J., Bayburt, E. K., Capparelli, M. P., Carroll, B. J., Goldstein, R. & Justice, M. R. et al. (1997). Discovery of CGS 27023A, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits. J. Med. Chem. 40, 2525-2532. Jeng, A. Y., Chou, M. & Parker, D. T. (1998). Sulfonamide-based hydroxamic acids as potent inhibitors of mouse macrophage metalloelastase. Bioorg. Med. Chem. Letters, 8, 897-902. Moy, F. J., Chanda, P. K., Chen, J. M., Cosmi, S., Edris, W., Skotnicki, J. S. et al. (1999). NMR solution structure of the catalytic fragment of human ®broblast collagenase complexed with a sulfonamide derivative of a hydroxamic acid compound. Biochemistry, 38, 7085-7096. Li, Y. C., Zhang, X., Melton, R., Ganu, V. & Gonnella, N. C. (1998). Solution structure of the catalytic domain of human stromelysin-1 complexed to a potent, nonpeptidic inhibitor. Biochemistry, 37, 14048-14056. Schechter, I. & Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157-162. Borkakoti, N. (1999). The structural basis of matrix metalloproteinase inhibition. Curr. Opin. Drug Disc. Dev. 2, 449-462. Shapiro, S. D., Grif®n, G. L., Gilbert, D. J., Jenkins, N. A., Copeland, N. G. & Welgus, H. G. et al. (1992). Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase. J. Biol. Chem. 267, 4664-4671. Jeng, A. Y., Wong, M., Duelfer, T., Shapiro, S. D., Kramer, R. A. & Hu, S. (1995). Mouse macrophage metalloelastase expressed in bacteria absolutely requires zinc for activity. J. Biochem. (Tokyo), 117, 216-221.
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Crystal Structure of MMP-12 30. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. 31. Gewirth, D. (1994). The HKL Manual, 3rd edit., Yale University , New Haven, CT. 32. Navaza, J. (1994). AMoRe: an automated package for molecular replacement. Acta Crystallog. sect. A, 50, 157-163. 33. Turk, D. (1992). Weiterentwicklung eines Programms fuer Molekuelgraphik und Elektronendichte
Manipulation und seine Anwendung auf verschiedene Protein-Strukturaufklaerungen. PhD thesis, TU Muenchen, Muenchen. 34. BruÈnger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P. & Grosse-Kunstleve, R. W. et al., (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905-921.
Edited by I. Wilson (Received 18 May 2001; received in revised form 12 July 2001; accepted 24 July 2001)
J. Mol. Biol. (2001) 312, 743±751
Crystal Structure of Human Macrophage Elastase (MMP-12) in Complex with a Hydroxamic Acid Inhibitor Herbert Nar1*, Karlheinz Werle2, Margit M. T. Bauer1, Horst Dollinger1 and Birgit Jung3 1
Department of Medicinal Chemistry, Boehringer Ingelheim Pharma KG Biberach, Germany 2
SibTeq Stable Isotope Biotechnique GmbH, Munich Germany 3 Department of Pulmonary Disease, Boehringer Ingelheim Pharma KG, Ingelheim Germany
Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in in¯ammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S10 -speci®city pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open speci®city pockets. The two crystallographically independent molecules adopt different conformations of the S10 -loop and its neighbouring loop due to differing crystal packing environments, suggesting that ¯exibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with b-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or ®ll the upper part of the speci®city pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12. # 2001 Academic Press
*Corresponding author
Keywords: macrophage metalloelastase; matrix metalloproteinase; MMP-12; crystal structure; chronic obstructive pulmonary disease
Introduction Human macrophage metalloelastase (MMP-12) is a member of a subclass of the zinc proteinase family of the metzincins,1 ± 3 the matrix metalloproteinases (MMPs), which degrade components of the extracellular matrix (ECM) in processes like Abbreviations used: MMP-12, human macrophage elastase; MMP, matrix metalloproteinase; ECM, extracellular matrix. E-mail address of the corresponding author: [email protected] 0022-2836/01/040743±9 $35.00/0
embryonic development, reproduction, cellular migration and tissue remodelling.1 MMP-12 is active against a broad range of ECM components, including elastin, ®bronectin, collagen IV, laminin, entactin, chondroitin sulfate and heparan sulfate. MMP-12 degrades a1-antitrypsin, which is the major inhibitor of human neutrophil elastase, thereby enhancing the overall elastolytic activity. Further, MMP-12 contributes to the angiostatin generation.4,5 In adult tissue, macrophages are the major source of MMP-12. Several studies have demonstrated that MMP-12 is involved in in¯ammatory processes and contributes to tissue remodelling # 2001 Academic Press
744 and tissue destruction. In tissue samples of colitis ulcerosa, MMP-12 was found abundantly expressed in macrophages in the in¯amed area and in the vicinity of shedding mucosal epithelium, supporting a role of this enzyme for tissue invasion and epithelial cell shedding.6 MMP-12 also contributes to the remodelling of elastolytic areas. A prominent expression of MMP-12 was detected in macrophages invading the atherosclerotic lesions induced in cholesterol-fed rabbits.7 In abdominal aortic aneurysms, a high expression of MMP-12 was demonstrated in macrophages within the degenerating aortic media.8 Abundant expression of MMP-12 was found in the skin after ultraviolet photoprovocation in areas of elastolytic material.9 Further insights into the role of MMP-12 were provided by studies with MMP-12-de®cient mice. Macrophages from MMP-12-de®cient mice were unable to penetrate basement membranes.10 In addition, these animals were protected from developing lung emphysema after chronic cigarette smoke exposure, indicating that MMP-12 is required for extracellular matrix proteolysis and tissue invasion, and plays an important role in mice in lung tissue destruction.11 MMP-12 shares common structural domains with other MMPs. The most closely related human matrix metalloproteinases are stromelysin-1 (MMP3) and interstitial collagenase (MMP-1), each with 49 % identity with MMP-12. The molecular mass of the MMP-12 proenzyme is 54 kDa and comprises three domains. The N-terminal proenzyme domain I (9 kDa short signaling peptide) includes a highly conserved cysteine residue that coordinates the zinc in the proenzyme forms.12,13 Domain II is the catalytic domain (22 kDa) that contains the zinc-binding HExxHxxGxxH sequence motif.14,15 This domain is followed by the ``hemopexin-like'' C-terminal domain III (23 kDa), which exhibits sequence homology to vitronectin and hemopexin. The 54 kDa MMP-12 proenzyme is activated, like all other matrix metalloproteinases, with loss of the N-terminal prosequence, leaving an active 45 kDa enzyme. This active MMP-12 is then readily processed to a mature 22 kDa species with loss of the C-terminal hemopexin-like domain.16 The structures of the catalytic domain of several members of the MMP family have recently become available (for a review, see Bode et al.15). A structural comparison reveals that the S10 subsite is the best-de®ned pocket in these MMPs and consists of a hydrophobic pocket that varies greatly in its depth. MMP-1 and MMP-7, which have shallow S10 pockets, prefer small hydrophobic amino acids at the P10 position. In contrast, MMP-3 and MMP8, which have deep S10 pockets, can accommodate large and small P10 amino acids with similar ef®ciency.17 MMP-12 has a preference for amino acids with large aliphatic side-chains, particularly leucine in the P10 position. Aromatic or hydrophobic amino acids are preferred at the P1 site, with small hydro-
Crystal Structure of MMP-12
phobic residues (preferably alanine) occupying P3.18 Because of its obvious functions in lung pathobiology, MMP-12 is an interesting pharmaceutical target. Therefore, we initiated a research program to determine the 3D structure of MMP-12 to enable us to study its interaction with ligands for the design of potent and selective inhibitors.
Results and Discussion We report the X-ray crystal structure of MMP-12 in complex with a hydroxamic acid inhibitor. The protein structure consists of the catalytic domain of MMP-12 (residues 100-280 of the complete sequence of the macrophage metalloelastase precursor, EC 3.4.24.65, Swissprot P39900) with an N-terminal glycine residue as a cloning artefact. This sequence was expressed in Escherichia coli as inclusion bodies and refolded into its active state. We observed partial autolytic cleavage of MMP-12 during the refolding and puri®cation steps at residue L250 (data not shown). Using the protocol described in Materials and Methods, the puri®ed protein contained only minor amounts of the C-terminally truncated lower molecular mass contaminant. The protein crystals that were grown from this material consist of pure, catalytically active wild-type enzyme. MMP-12 crystallises in the orthorhombic space group I222 with two molecules in the asymmetric unit. The structure was solved by molecular replacement using a homology model that was based on the 3D structure of stromelysin-1 (MMP-3, PDB Ê resolution code 1hfs) and was re®ned to 2.6 A with good stereochemistry and crystallographic R-factors (Table 1). Apart from the 16 C-terminal residues (residue numbers 265-280), which are not visible and probably dynamically disordered, the molecule is completely de®ned in the electron density (Figure 1). The two independent molecules were kept very similar during re®nement, apart from residues 204214 and 240-249, which are in different crystalline environments and were re®ned independently. In the crystal lattice, the molecules pack with their catalytic clefts oriented to one another so that two symmetry equivalent inhibitor molecules are in close contact. Molecular structure of MMP-12 As expected, MMP-12 adopts a fold that is highly similar to the other Zn-proteinases of the MMP protein family consisting of a ®ve-stranded b-sheet and three a-helices (Figure 2). The catalytic zinc ion is ligated by three histidine residues from the sequence motif HExxHxxGxxH at the C-terminal end of the central helix hB and the turn following it. b-Strand sIV functions as the template with which substrate peptide are associated in a functional complex via main-chain hydrogen bonding. In addition to the catalytic zinc ion, there is a struc-
Crystal Structure of MMP-12
745
Figure 1. Stereo representation of the active site with the zinc ligation sphere, the template strand residues and bound inhibitor superimposed on the experimental 2Fo ÿ Fc omit map contoured at 1.2s.
tural zinc ion and three calcium ions bound by the polypeptide chain. The loop following the Met-turn structure in the MMPs1 circumscribes the S10 -subsite that is the most prominent feature of the catalytic site because it forms a deep pocket and is the main speci®city determinant of the MMPs. The S10 -loop structure in MMP-12 (residues 240-250) is well ordered in both crystallographically independent molecules in the crystal lattice. Residues D244 to F248 at the Cterminal end of the S10 -loop form an a-helical turn structure similar to the analogous region in MMP8. It is stabilized mainly by hydrophobic interactions of residues V243, I245 and F248 with F213 and L214 of the neighbouring loop and a hydrogen bond formed by the strictly conserved Y240 OH with the backbone NH moiety of N211 (Figure 3). A different crystal packing environment for the two independent molecules induces a slightly different position of the C-terminal end of the loop Ê shift and a dis(residues 243-249) comprising a 1 A torsion of the helical turn in molecule A. B-factors indicate a higher than average mobility of the Cterminal part of the S10 loop in molecule B, which faces a solvent channel in the lattice, while this is not the case for molecule A, probably because here crystal contacts to a symmetry-related molecule stabilize a more rigid structure of the loop. Structural rearrangements upon ligand binding19 as well as S10 loop mobility are detected in X-ray and dynamic NMR-studies of MMP-13.20 The above observations of ¯exibility or adjustment of this part of the S10 -loop are consistently seen in other MMPstructures and are therefore probably a common feature for all MMPs. The sequence stretch of residues 204-214 between secondary structural elements sV and hB is located next to the S10 -loop and forms hydrophobic and hydrogen bond interactions with it. Its lower end exhibits stuctural differences in molecules A and B about residue H206, again
induced by different crystal packing environments. The key residue for the structuring of this loop, T204, forms hydrogen bonds to the carbonyl groups of H206 and G208 in molecule B, but only to H206 CO in molecule A, because here the mainchain conformation of residue G208 directs its carbonyl group into bulk solvent. The side-chain of H206 is in two conformations, differing in the w1-torsion angle, which is energetically more favourable in molecule A, whereas in molecule B the unfavourable conformation is compensated by the concomitant formation of a hydrogen bond of neighbouring T205 CO to K148 Nx of a symmetryrelated molecule. The N terminus of the expressed construct of the MMP-12 catalytic domain, residues F100 REMP104, preceded by a glycine residue that originates from the used expression vector, is a wellordered structure that is main-chain hydrogen bonded to a sequence stretch around the zinc-ligating H228 and forms a short, two-stranded antiparallel b-sheet. F100 NH is bound to the carboxylate group of D253, the ®rst of a conserved tandem of aspartate residues positioned in the ®rst turn of helix hC. This interaction is reminiscent of the interaction of the charged N terminus of a ``superactivated'' form of neutrophil collagenase (MMP8).21 The N-terminal glycine residue is within H-bonding distance from S230 OH. The ordered structure of the MMP-12 N terminus is in fact identical with the N termini of the F79-form of MMP-8 (PDB code 1kbc) and of matrilysin (PDB code 1mmq). It may represent the structure of the mature enzyme and may be more active than any N-terminally truncated forms. Residue M103, which is unique to MMP-12, is thereby positioned next to H222 and forms, together with residues P104 GGP107 and G186 on strand sIV, a major part of the unprimed side of the active-site cleft.
746
Crystal Structure of MMP-12
respectively), for MMPs 3, 8 and 13 this residue is leucine and the pocket is much larger. In MMP-12, this respective residue is leucine (L214). The conformation of the MMP-12 S10 -loop is very similar to the loop of MMP-8 especially because both proteins exhibit one helical turn at residues D244-F248 (MMP-12 sequence numbers) at the bottom end of the S10 -subsite. However, the MMP-8 S10 -subsite is closed at the far side due to the presence of an arginine residue (R222, V243 in MMP-12), while in MMP-12 the S10 -pocket extends into a channel that reaches through the protein to the far side of the protein surface. Thus, with respect to the 3D shape of the S10 -pocket, MMP-12 more closely resembles MMPs-3 and 13 in having a large open speci®city pocket. Inhibitor-protein interactions
Figure 2. Molecular structure of MMP-12 in complex with CGS27023A. (a) Ribbon representation of the catalytic domain structure with the bound metal ions, which are coloured blue (zinc) and grey (calcium). (b) Surface representation.
Comparison to other MMPs A comparison of the structurally characterized members of the MMP family reveals a topologically homogeneous ensemble of 3D structures with almost identical positions of secondary structural elements and loops (Figure 4). The largest deviations occur in the S10 -loop segment and the neighbouring loop, regions that are sequentially most divergent. The size of the S10 -pocket is different in the various MMPs because of the sequence variability and length of the S10 -loop and the type of amino acid positioned four residues N-terminal to the ®rst zinc-ligating histidine of the HExxHxxGxxH motif.19 While for MMP-1 and MMP-7 the pocket is small and closed due to the presence of a large residue at this position (arginine or tyrosine,
The hyroxamic acid inhibitor CGS27023A was originally discovered as an orally active stromelysin-1 (MMP-3) inhibitor that ef®ciently blocks cartilage degradation.22 Its af®nity toward mouse MMP-12 was recognized recently.23 This compound is a potent inhibitor of various members of the MMP family exhibiting only weak selectivity against MMPs 1, 2, 7, 8, 9, 12 and 13 (IC50 values 49.5 nM, 9.1 nM, 16.9 nM, 106 nM, 4.4 nM, 4.3 nM, 2.0 nM, 4.3 nM respectively). 3D solution structures of MMPs 1, 3 and 13 with CGS27023A have been reported based on NMR data.20,24,25 In the present structure, CGS27023A is bound to the catalytic zinc ion, as expected in a bidentate fashion, by the two hydroxamate oxygen atoms, resulting in a trigonal bipyramidal coordination of the zinc ion (Figure 5). The remainder of the ligand occupies the primed side26 of the active-site cleft. The protonated hydroxamate oxygen forms an asymmetric bifurcated hydrogen bond with the side-chain carboxylate group of E219. The hydroxamate NH group is involved in a hydrogen bond with A182 and the sulfone group forms a hydrogen bond with one of its oxygen atoms to L181 NH, mimicking one of the canonical hydrogen bonds formed by peptide substrates of the enzyme with these b-strand IV residues. The other potential hydrogen bonding groups of the inhibitor face bulk solvent. The isopropyl substituent does not form any interaction with the protein and is mainly solventexposed, forming solely intramolecular contacts to the edge of the pyridyl ring. The pyridyl subtituent sticks out of the active-site cleft above P238 and forms a close contact of its o-CH group with the carbonyl group of the same residue, another example of a hydrogen bond formed by an aromatic ring system. The methoxyphenyl moiety is placed in the upper part of the S10 -pocket of the proteinase between residues L181, T215 and the zinc-ligating H218. The lower part of the pocket is left unoccupied by the inhibitor and is ®lled with an array of water molecules. As mentioned above,
Crystal Structure of MMP-12
747
Figure 3. Stereo representation of the structure of the catalytic site with bound ligand and the S10 -loop forming the speci®city pocket of the enzyme. CGS27023A reaches only halfway into the large cavity, which is otherwise ®lled by solvent.
the S10 -pocket is in fact a channel that connects the active-site cleft with the other side of the protein so that the observed ordered water molecules are in rapid exchange with bulk sovent through this channel. The bound conformation of the inhibitor is a low-energy conformation in which the torsion angles about the rotatable bonds are close to stereochemically ideal values. The observed binding mode of CGS27023A reveals that a favourable bound conformation, strong hydrogen bonding and hydrophobic interactions contribute to the strong af®nity for MMP12 (IC50 2 nM). On the other hand, all interactions described can be formed equally well with various
other members of the MMP-family, which in turn explains the missing or weak selectivity pro®le of CGS27023A. In the present structure, a major crystal contact is formed by the interaction of the active-site clefts of two symmetry-related molecules. The contacts made by the protein are exclusively polar, namely hydrogen bonds of H228 NdH to D175 OD, R101 NH1 to K177 CO and F171 CO to H172 NdH, all of which follow dyad symmetry and are therefore repeated twice for a given pair of MMP-12 dimers. Thereby, the pyridyl and isopropyl substituents of the two inhibitor molecules that occupy both active sites come into close contact. In fact, the pyridyl
Figure 4. Stereo representation of a superposition of the backbone ribbons of MMP-1 (PDB code 1HFC, magenta), MMP-2 (PDB code 1CK7, blue), MMP-3 (PDB code 1SLM, light blue), MMP-7 (PDB code 1MMQ, green), MMP-8 (PDB code 1JAP, yellow), MMP-13 (PDB code 966C, orange) with MMP-12 (red). The secondary structural elements superimpose almost perfectly. Structural diversity is most prominent in the S10 -loop region and the neighbouring loop (lower right) and at the N terminus (left).
748
Crystal Structure of MMP-12
large P10 -groups, which convey differences in inhibition of about one to three orders of magnitude for MMP-3, 8 and 13 (large open S10 -pockets) versus MMP-1 and 7 (smaller S10 -pockets). However, binding of inhibitors with large P10 -groups is not generally prohibitive, as shown for MMP-1, for which an induced ®t of the enzyme is found when it is complexed with such an inhibitor.19 This study shows that MMP-12 falls into the category of MMPs 8 and 13 with a large S10 -pocket. Any design of ligands with large P10 -subtituents should therefore yield selectivity towards the other group of MMPs. A distinction within the ®rst group, however, will have to be made using subtituents that bind to other subsites of the enzyme.
Materials and Methods Chemical synthesis of CGS27023A The synthesis of the MMP inhibitor CGS27023A was as described.22 Molecular cloning of the proteinase domain of MMP12
Figure 5. Enzyme-inhibitor interactions. (a) Close-up view of the active site and into the S10 -speci®city pocket of MMP-12, which reaches through the protein and opens up to the other side. (b) A representation of the interactions of CGS27023A with the enzyme.
rings are in a perfect aromatic stacking arrangeÊ . From ment with an inter-ring distance of 3.5 A this observation it may be inferred that the bound conformation of the ligand is perturbed by the described crystal contact. A comparison with the observed conformations of the inhibitor bound to the other MMPs in the NMR solution structures, however, shows very similar overall features. Based on the observed low-energy conformation of the inhibitor and the similarity to the solution structures of CGS27023A complexed to MMP-1, 3 and 13, we reason that the crystal packing forces do not perturb the bound inhibitor structure signi®cantly in the present structure. Implications for drug design and selectivity The potency of almost all reported MMP inhibitors relies strongly on a zinc-binding functionality, substrate-like hydrogen bonding along b-stand sIV and hydrophobic interactions made in the the S10 pocket. Most other subsites are shallow and exposed to solvent and thus not readily used for increasing af®nity.27 The design of selectivity of inhibitors of MMPs consequently is based mainly on the in¯uence of
The domain II (proteinase domain) of the human macrophage elastase 12 (amino acid residues 100-279) was ampli®ed by PCR with human-speci®c primers hMP1 (33mer): 50 -AAACCATGGGTTTCAGGGAAATGC CAGGGGGGC-30 and hMP2 (38mer): 50 -AAAGGATCCTATTAAGCTGGTTCTGAATTGTCAGGATT-30 from a human placental cDNA bank (Clonetech HL1075b) and cloned in the NcoI/BamHI restriction site of the multiple cloning site of the expression vector pET8C (Novagen) as described.28,29 The insert (567 bp) was fully sequenced for control and the plasmid pHME transformed to E. coli BL21 (DE3) pLysS (Novagen). Expression The cells were grown in a 7.5 l fermenter (BioFlo IV, New Brunswick Scienti®c) under online control of pH, O2 and temperature in slightly modi®ed commercial minimal medium M9 (Sigma, Deisenhofen). At an A600 of 1, the expression was induced by adding IPTG to 1 mM. At 12 hours after induction the cells were pelleted by centrifugation at 3000 g for 30 minutes and frozen (ÿ70 C). Purification The cells were resuspended in 1000 ml of lysis buffer (50 mM Tris-HCl, pH 7.4) and disrupted by sonication and incubation with lysozyme for one hour at 37 C. After three extensive washing steps with lysis buffer and lysis buffer containing 0.05 % (w/v) Brij 35 and 1 M urea, the inclusion bodies pellet was resuspended in 1000 ml of lysis buffer containing 8 M urea and incubated for one hour at 37 C. Insoluble material was removed by centrifugation at 50,000 g and the supernatant was dialyzed in four steps against 15 l of dialysis buffer (50 mM Tris, 30 mM NaCl, 10 mM CaCl2, 0.05 % Brij 35) with decreasing concentration of urea. Precipitated protein was removed by centrifugation at 8000 g for 30 minutes. The supernatant was loaded onto
749
Crystal Structure of MMP-12 Table 1. Data collection and re®nement statistics Space group I222 Ê) Unit cell dimensions (A a 67.4, b 87.2, c 169.2 Ê) Resolution limits (A 100.0-2.6 Total observations 57,864 Unique reflections 15,776 Completeness (%) Overall 100 Ê ) shell (2.60-2.55 A 100 Rmerge Overall 0.18 Ê shell 2.60-2.55 A 0.58 Protein atoms, water molecules, inhibitor atoms, metal ions 2606, 333, 54, 10 R factor (%) 19.6 25.7 Rfree Ê) Resolution range (A 20.0-2.6 Number of reflections (free set) 15,757 (797) Rms deviation from ideal geometry Ê) Bond length (A 0.006 Bond angles (deg.) 1.17 2 Ê ) Temperature factors (A B-value model Restrained individual B-factors All protein atoms 27.1 rms bonded B 0.8
a column containing the tripeptide hydroxamate H-ProLeu-Gly-NHOH (Bachem) coupled to CH-Sepharose 4B (Pharmacia) and the matrix was washed with 50 mM Tris (pH 7.0), 500 mM NaCl until no more protein could be detected in the ¯ow-through. The MMP12 was eluted with elution buffer (50 mM Tris (pH 11), 500 mM NaCl) into a vessel containing about one-tenth of the elution volume of 1 M Tris (pH 7.0). Crystallization The eluted protein was inhibited with CGS27023A, subsequently the protein buffer was exchanged with the help of PD10-columns (Pharmacia) against the crystallization buffer (20 mM Tris (pH 7.4), 200 mM NaCl, 2 mm CaCl2). After concentrating the protein sample to 4 mg/ ml, crystallization was carried out with the hanging drop vapour diffusion method in Q-plates (Hampton research). Hexagonal-shaped crystals up to a size of 0.2 mm 0.2 mm 0.1 mm were grown after four to ten days at 4 and 20 C with a reservoir containing 100 mM sodium-cacodylate (pH 6.5) and 1.4 M sodium acetate. Data collection Data were collected from cryo-cooled crystals which were cryo-protected by addition of 30 % (v/v) glycerol to the mother liquor. Two native data sets were collected from two different crystal specimens on our in-house rotating anode generator with Cu-Ka radiation and on the synchrotron wiggler Ê . The latter beamline 6B at DESY at wavelength 1.05 A Ê resolution was used for data set extending to 2.6 A re®nement of the structure. The crystal space group is I222 with cell dimensions of Ê , b 87.2 A Ê , c 169.2 A Ê with two crystallograa 67.4 A phically independent molecules in the asymmetric unit. Using a molecular mass of 21 kDa for the expressed version of the MMP-12 catalytic domain, the speci®c Ê 3/Da and the approxivolume (VM) calculated is 3.0 A
mate solvent content is 54 % (v/v).30 Data were integrated, scaled and merged with the HKL package.31 Molecular replacement The structure was solved using AMoRe32 with a homology model of MMP-12 derived from the crystal structure of stromelysin (PDB code 1HFS) which displays 49 % sequence homology to MMP-12. The two molecules could be placed in the asymmetric unit unambiguously. They are related to each other by an improper local symmetry with a rotation component of 50 about the crystallographic c-axis. Model building and refinement The ®rst electron density was computed with the homology model used for molecular replacement. Density averaging over the two independent molecules combined with solvent ¯ipping was performed using MAIN.33 The electron density at this stage was interpretable for most of the protein chain. Repeated cycles of model building, rephasing and averaging were performed until a complete structural model was obtained. Water molecules were added based on peaks in difference Fourier maps and geometric considerations. The model was re®ned using CNS34 using data Ê resolution. Restrained non-crysbetween 20.0 and 2.6 A tallographic symmetry was used throughout re®nement. In later stages, deviations between the two molecules were allowed in the sequence regions 204-214 and 240249, which have slightly different main chain positions and distinct side-chain conformations due to crystal packing effects. The re®ned MMP-12 model has an R-value of 19.6 % and an Rfree value of 25.7 %. The r.m.s. deviation of the Ê (0.11 A Ê for Ca atoms two copies for all atoms is 0.29 A leaving out the regions 204-214 and 240-249). Data collection, re®nement statistics and geometrical parameters for the ®nal model are presented in Table 1. Protein Data Bank accession code The coordinates and structure factors for the present MMP-12-CGS27023A complex were deposited in the Protein Data Bank, accession code 1JIZ.
Acknowledgements We thank Professor Walter StoÈcker, University of MuÈnster, Germany, for the af®nity puri®cation protocol and helpful discussions, Dr Thomas Fox for providing us with a homology model for MMP-12 and Rebecca Scherer and Andrea KuÈhn for technical support. We acknowledge the help of Hans Bartunik during data collection on the MPG/GBF wiggler beamline BW6/DORIS at DESY.
References 1. Bode, W., Gomis-RuÈth, F. X. & StoÈcker, W. (1993). Astacins, serralysins, snake venom and matrix metalloproteinases exhibit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) and
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Edited by I. Wilson (Received 18 May 2001; received in revised form 12 July 2001; accepted 24 July 2001)