Portal de Periódicos da CAPES

The 1.8-Å Crystal Structure of a Matrix Metalloproteinase 8-Barbiturate Inhibitor Complex Reveals a Previously Unobserved Mechanism for Collagenase Substrate Recognition

2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês

10.1074/jbc.m007475200

1083-351X

Hans Brandstetter, Frank Grams, Dagmar Glitz, A. Lang, Robert Huber, Wolfram Bode, Hans‐Willi Krell, Richard A. Engh,

Blood Coagulation and Thrombosis Mechanisms

The individual zinc endoproteinases of the tissue degrading matrix metalloproteinase (MMP) family share a common catalytic architecture but are differentiated with respect to substrate specificity, localization, and activation. Variation in domain structure and more subtle structural differences control their characteristic specificity profiles for substrates from among four distinct classes (Nagase, H., and Woessner, J. F. J. (1999)J. Biol. Chem. 274, 21491–21494). Exploitation of these differences may be decisive for the design of anticancer or other drugs, which should be highly selective for their particular MMP targets. Based on the 1.8-Å crystal structure of human neutrophil collagenase (MMP-8) in complex with an active site-directed inhibitor (RO200-1770), we identify and describe new structural determinants for substrate and inhibitor recognition in addition to the primary substrate recognition sites. RO200-1770 induces a major rearrangement at a position relevant to substrate recognition near the MMP-8 active site (Ala206–Asn218). In stromelysin (MMP-3), competing stabilizing interactions at the analogous segment hinder a similar rearrangement, consistent with kinetic profiling of several MMPs. Despite the apparent dissimilarity of the inhibitors, the central 2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor RO200-1770 mimics the interactions of the hydroxamate-derived inhibitor batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T., Pieper, M., Tschesche, H., Huber, R., and Bode, W. (1995)Eur. J. Biochem. 228, 830–841) for binding to MMP-8. The two additional phenyl and piperidyl ring substituents of the inhibitor bind into the S1′ and S2′ pockets of MMP-8, respectively. The crystal lattice contains a hydrogen bond between the Oγgroup of Ser209 and Nδ1 of His207 of a symmetry related molecule; this interaction suggests a model for recognition of hydroxyprolines present in physiological substrates. We also identify a collagenase-characteristic cis-peptide bond, Asn188–Tyr189, on a loop essential for collagenolytic activity. The sequence conservation pattern at this position marks this cis-peptide bond as a determinant for triple-helical collagen recognition and processing. The individual zinc endoproteinases of the tissue degrading matrix metalloproteinase (MMP) family share a common catalytic architecture but are differentiated with respect to substrate specificity, localization, and activation. Variation in domain structure and more subtle structural differences control their characteristic specificity profiles for substrates from among four distinct classes (Nagase, H., and Woessner, J. F. J. (1999)J. Biol. Chem. 274, 21491–21494). Exploitation of these differences may be decisive for the design of anticancer or other drugs, which should be highly selective for their particular MMP targets. Based on the 1.8-Å crystal structure of human neutrophil collagenase (MMP-8) in complex with an active site-directed inhibitor (RO200-1770), we identify and describe new structural determinants for substrate and inhibitor recognition in addition to the primary substrate recognition sites. RO200-1770 induces a major rearrangement at a position relevant to substrate recognition near the MMP-8 active site (Ala206–Asn218). In stromelysin (MMP-3), competing stabilizing interactions at the analogous segment hinder a similar rearrangement, consistent with kinetic profiling of several MMPs. Despite the apparent dissimilarity of the inhibitors, the central 2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor RO200-1770 mimics the interactions of the hydroxamate-derived inhibitor batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T., Pieper, M., Tschesche, H., Huber, R., and Bode, W. (1995)Eur. J. Biochem. 228, 830–841) for binding to MMP-8. The two additional phenyl and piperidyl ring substituents of the inhibitor bind into the S1′ and S2′ pockets of MMP-8, respectively. The crystal lattice contains a hydrogen bond between the Oγgroup of Ser209 and Nδ1 of His207 of a symmetry related molecule; this interaction suggests a model for recognition of hydroxyprolines present in physiological substrates. We also identify a collagenase-characteristic cis-peptide bond, Asn188–Tyr189, on a loop essential for collagenolytic activity. The sequence conservation pattern at this position marks this cis-peptide bond as a determinant for triple-helical collagen recognition and processing. matrix metalloproteinase tumor necrosis factor α converting enzyme The matrix metalloproteinases (MMPs),1 one of the five families that form the metzincin group of zinc proteinases (3Stöcker W. Grams F. Baumann U. Reinemer P. Gomis-Rüth F.X. McKay D.B. Bode W. Protein Sci. 1995; 4: 823-840Crossref PubMed Scopus (633) Google Scholar), function to degrade the extracellular matrix during embryonic development, reproduction, and tissue remodeling (1Nagase H. Woessner J.F.J. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar) but are disregulated in arthritis, cancer, and other diseases. The "minimal" MMPs matrilysin and endometase (MMP-7 and MMP-26, respectively), have a Zn2+ and Ca2+ binding catalytic domain, and an N-terminal pro-domain. All other known MMPs possess additionally a hemopexin-like domain near the C terminus, and further domain insertions differentiate MMP subfamilies. Gelatinases A and B (MMP-2 and MMP-9) possess three fibronectin type II-like repeats inserted at a loop in the catalytic domain; these form an independent folding domain adjacent to the catalytic domain. Membrane-type MMPs possess an anchoring transmembrane helix C-terminal to the hemopexin-like domain (4Murphy G. Knäuper V. Matrix Biol. 1997; 15: 511-518Crossref PubMed Scopus (260) Google Scholar). Hierarchical regulation of MMP activity occurs on many levels, including gene expression control (1Nagase H. Woessner J.F.J. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar, 5Westmays J.A. Strissel K.J. Sadow P.M. Fini M.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6768-6772Crossref PubMed Scopus (92) Google Scholar), proteolytic activation of MMP zymogens (6van Wart H. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1197) Google Scholar), inhibition by endogenous tissue inhibitors of metalloproteinases (7Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar), and both positive and negative proteolytic feedback loops (8Patterson B.C. Sang Q.X.A. J. Biol. Chem. 1997; 272: 28823-28825Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 9Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1131) Google Scholar). Crystal structures of several MMPs have been determined (for a review, see, e.g., Ref. 10Bode W. Fernandez-Catalan C. Tschesche H. Grams F. Nagase H. Maskos K. Cell. Mol. Life Sci. 1999; 55: 639-652Crossref PubMed Scopus (298) Google Scholar), revealing overall domain structures, catalytic mechanisms, and many aspects of MMP regulation mechanisms; these include collagenase 1 (MMP-1) (11Li J. Brick P. O'Hare M.C. Skarzynski T. Lloyd L.F. Curry V.A. Clark I.M. Bigg H.F. Hazleman B.L. Cawston T.E. Blow D.M. Structure. 1995; 3: 541-549Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 12Lovejoy B. Welch A.R. Carr S. Luong C. Broka C. Hendricks R.T. Campbell J.A. Walker K.A.M. Martin R. Van Wart H. Browner M.F. Nat. Struct. Biol. 1999; 6: 217-221Crossref PubMed Scopus (255) Google Scholar) and collagenase 2 (MMP-8). Structures of the latter are represented by two forms of the catalytic domain, resulting from activation cleavage alternately at two cleavage sites, leaving either Met80 (13Stams T. Spurlino J.C. Smith D.L. Wahl R.C. Ho T.F. Qoronfleh M.W. Banks T.M. Rubin B. Nat. Struct. Biol. 1994; 1: 119-123Crossref PubMed Scopus (201) Google Scholar, 14Bode W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (323) Google Scholar) or Phe79 as the N-terminal residue (15Reinemer P. Grams F. Huber R. Kleine T. Schnierer S. Piper M. Tschesche H. Bode W. FEBS Lett. 1994; 338: 227-233Crossref PubMed Scopus (136) Google Scholar). The latter form is "superactivated," as Phe79 forms a salt bridge with Asp232 and thereby prevents the N-terminal sequence from transient or other interference with the active site. The result is a 3-fold increase in activity compared with activation cleavage at Met80(16Knäuper V. Wilhelm S.M. Seperack P.K. DeClerck Y.A. Langley K.E. Osthues A. Tschesche H. Biochem. J. 1993; 295: 581-586Crossref PubMed Scopus (154) Google Scholar). As their early nomenclature implies, collagenases I, -II, and -III (17Knäuper V. Lopezotin C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar) (MMP-1, -8, and -13, respectively) degrade mainly fibrillar collagens (18Netzel-Arnett S. Fields G.B. Birkedal-Hansen H. Van Wart H.E. J. Biol. Chem. 1991; 266: 6747-6755Abstract Full Text PDF PubMed Google Scholar, 19Ottl J. Battistuta R. Pieper M. Tschesche H. Bode W. Kuhn K. Moroder L. FEBS Lett. 1996; 398: 31-36Crossref PubMed Scopus (82) Google Scholar, 20Knäuper V. Cowell S. Smith B. Lopezotin C. Oshea M. Morris H. Zardi L. Murphy G. J. Biol. Chem. 1997; 272: 7608-7616Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar), although the structural origin of this specificity is not well understood (4Murphy G. Knäuper V. Matrix Biol. 1997; 15: 511-518Crossref PubMed Scopus (260) Google Scholar). Disruption of MMP tissue remodeling function causes a variety of disorders, including cancer (tumor growth, invasion and metastasis), rheumatoid arthritis and osteoarthritis, and a variety of diseases involving neovascularization. The resulting clinical need has fostered an enormous interest in the development of inhibitors against MMPs. As part of these efforts, crystal structures of MMPs with a variety of synthetic inhibitors have been determined. For MMP-8, complexes reported include peptide mimetics, hydroxamic acid derivatives (2Grams F. Reinemer P. Powers J.C. Kleine T. Pieper M. Tschesche H. Huber R. Bode W. Eur. J. Biochem. 1995; 228: 830-841Crossref PubMed Scopus (178) Google Scholar, 14Bode W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (323) Google Scholar, 21Betz M. Huxley P. Davies S.J. Mushtaq Y. Pieper M. Tschesche H. Bode W. Gomis-Rüth F.X. Eur. J. Biochem. 1997; 247: 356-363Crossref PubMed Scopus (88) Google Scholar, 22Dhanaraj V. Williams M.G. Ye Q.Z. Molina F. Johnson L.L. Ortwine D.F. Pavlovsky A. Rubin J.R. Skeean R.W. White A.D. Hubmlet C. Hupe D.J. Blundell T.L. Croat. Chem. Acta. 1999; 72: 575-591Google Scholar), phosphinamides and sulfodiimines (23Chen L.Y. Tydel T.J. Gu F. Dunaway C.M. Pikul S. Dunham K.M. Barnett B.L. J. Mol. Biol. 1999; 293: 545-557Crossref PubMed Scopus (71) Google Scholar, 24Browner M.F. Smith W.W. Castelhano A.L. Biochemistry. 1995; 34: 6602-6610Crossref PubMed Scopus (250) Google Scholar), thiadiazole (25Finzel B.C. Baldwin E.T. Bryant G.L. Hess G.F. Wilks J.W. Trepod C.M. Mott J.E. Marshall V.P. Petzold G.L. Poorman R.A. Osullivan T.J. Schostarez H.J. Mitchell M.A. Protein Sci. 1998; 7: 2118-2126Crossref PubMed Scopus (74) Google Scholar), and malonic acid derivatives (26Brandstetter H. Engh R.A. von Roedern E.G. Moroder L. Huber R. Bode W. Grams F. Protein Sci. 1998; 7: 1303-1309Crossref PubMed Scopus (47) Google Scholar, 27von Roedern E. Brandstetter H. Engh R.A. Bode W. Grams F. Moroder L. J. Med. Chem. 1998; 41: 3041-3047Crossref PubMed Scopus (13) Google Scholar). Here we describe the 1.8-Å crystal structure of MMP-8 inhibited by a barbituric acid derivative. Conformational rearrangements accompanying the inhibitor binding lead to a new and highly ordered crystal packing arrangement. The high resolution structural data enables a thorough analysis of determinants of MMP selectivity toward both low molecular weight substances as well as substrate classes. A previously unreported cis-peptide bond (Asn188—Tyr189) could be unambiguously identified. The conservation patterns of the sequence at the cis-peptide bond position support the hypothesis that this cis-peptide plays a critical role in substrate recognition mechanisms specific to the collagenases I and II (MMP-1 and MMP-8). MMP-1, MMP-3, and MMP-8 were kindly provided by Profs. G. Murphy (University of East Anglia, Norwich, United Kingdom), H. Nagase (Imperial College, London, United Kingdom), and H. Tschesche (University of Bielefeld, Bielefeld, Germany), respectively; MMP-2 and MMP-9 were obtained from Roche Molecular Biochemicals (Penzberg, Germany); MT1-MMP and MT3-MMP were provided by Prof. J. Foidart (Université de Liège, Liège, Belgium). The inhibitors RO200-1770, RO204-1924, I-COL043, RO206-0027, and RO206-0032 were synthesized as described previously (28Bosies E. Esswein A. Grams F. Krell H.W. New Barbituric Acid Derivative, Processes Forware Production, and Pharmaceutical Agents Containing these Compounds. Boehringer Mannheim, Indianapolis, IN1996Google Scholar). The fluorogenic substrate M-1855 (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-d-Arg-NH2) was purchased from Bachem (Heidelberg, Germany); all other chemicals were of highest purity commercially available. All measurements were performed at 25 °C using a buffering solution of 50 mm Tris, pH 7.6, 100 mm NaCl, 10 mm CaCl2. Based on multiple measurements, all data are precise to within 5%. Depending on activity, enzymes were used at 5–50 nm concentration range with a substrate concentration of 2.55 μm. The enzyme was briefly pre-incubated with the inhibitor at a resultant Me2SO concentration of 1%. The reaction was started with the addition of the substrate M-1855. Substrate was excited at 280 nm and the substrate fluorescence was monitored at 346 nm using the FuoroMax-3 fluorometer (SPEX, Horiba Group, Grasbrunn/Munich, Germany). MMP-8 was concentrated to 8 mg/ml and then mixed with 3-fold molar excess of an aqueous solution of RO200-1770 for a final MMP-8 concentration of 6 mg/ml. 3 μl of protein-inhibitor complex was mixed with 2 μl precipitant solution containing 100 mmcacodylate pH 5.5–6.5, 10 mm CaCl2, 100 mm NaCl, and 10% polyethylene glycol 6000. The hanging drop was equilibrated by vapor diffusion at room temperature against a reservoir containing 1.0–1.5 m phosphate buffer. Data were collected on a multiwire detector (X1000, Bruker AXS) to 1.8-Å resolution and processed using SAINT data reduction software (29Howard A.J. Gilliland G.L. Finzel B.C. Poulos T.L. Ohlendorf D.H. Salemme F.R. J. Appl. Crystallogr. 1987; 20: 383-387Crossref Scopus (571) Google Scholar), yielding an agreement of redundant measurements ofRmerge = 9.3% over all data and 41% in the outer resolution shell (completeness 98% and 87%, respectively). The space group of the crystal was determined as I222 with unit cell dimensions a = 61.02 Å, b = 69.24 Å, c = 88.47 Å. The orientation and translation of the molecule within the crystallographic unit cell was determined with Patterson search techniques (30Huber R. Acta Crystallogr. 1965; 19: 353-356Crossref Google Scholar, 31Hoppe W. Acta Crystallogr. 1957; 10: 750-751Google Scholar, 32Rossmann M.G. Blow D.M. Acta Crystallogr. 1962; 15: 24-31Crossref Google Scholar) using the program AMoRe (33Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar). Electron density calculation and model building proceeded using the program MAIN (34Turk D. Chemistry. Technische Universität München, Munich, Germany1992Google Scholar). The structure has been refined by using the program X-PLOR (35Brunger A.T. X-PLOR: A System for X-ray Crystallography and NMR , Version 3.1. Yale Univeristy Press, New Haven, CT1992Google Scholar) to a crystallographic R-value of 21.1% (Rfree = 29.6%) with bond deviations of 0.009 Å and angle deviations of 1.7° from ideality (36Engh R.A. Huber R. Acta Crystallogr. Sect. A. 1991; 47: 392-400Crossref Scopus (2539) Google Scholar). The molecular structure was analyzed and compared using appropriate tools within the program MAIN (34Turk D. Chemistry. Technische Universität München, Munich, Germany1992Google Scholar). The inhibitor RO200-1770 is a barbituric acid derivative, doubly substituted with phenyl and 4-ethanolpiperidyl rings as depicted in Fig.1. The barbiturate ring chelates the zinc and rigidly orients the two cyclic substituents into the S1′ and S2′ substrate binding sites. Neither substituent ring system appears strained by the protein environment, although their relative orientations may be induced by protein binding. The phenyl moiety occupies the MMP-8 binding site and is perfectly planar to within the 1.8-Å resolution. The electron density observed for the piperidine ring allows an interpretation whereby two chair conformations related by a 180° rotation along the C5-pN1 bond might be superimposed; either conformation would allow favorable hydrophobic contacts in the S2′-site. Adopting an all-trans conformation, the alcohol group points toward the solvent. The relative orientations of the rings of the inhibitor may be described by considering the ring planes and the bonds linking the substituent rings to the C5 atom of the barbiturate ring. The C5–fC1 bond linking the phenyl ring is nearly perpendicular to the plane of the barbiturate ring (excluding C5). This arrangement necessarily orients the plane of the phenyl ring likewise perpendicular to the barbiturate plane. The dihedral angle C6–C5–fC1–fC2 fixes the ring orientation with an eclipsed geometry (at 1.2°, while the C4–C5–C1–fC2dihedral is staggered at 60.8°). In contrast, the C5–pN1 bond lies nearly in the plane of the barbiturate, extending the P2′-piperidyl ring away from the barbiturate; all dihedrals across the C5—pN1bond have staggered orientations. This results in an arrangement where all three rings are mutually perpendicular, as follows: the angle between (the normal vectors) of the barbiturate and phenyl rings is 91°, between the barbiturate and the piperidyl rings is 103°, and between the phenyl and piperidyl rings is 111°. The MMP-8 substrate recognition sites are shown schematically in Fig. 3 A. Comparison with the binding mode of RO200-1770 as depicted in Fig. 3 Bhighlights the inhibitor binding at the "primed" substrate recognition sites and at the Zn2+ ion. The Zn2+is coordinated by atoms N3 and O2 of the barbiturate ring. The Zn2+—N3 coordination has a favorable distance of 2.09 Å and highly symmetric Zn2+-N3-C2 and Zn2+-N3-C4 angles of 119° and 117°, respectively. Positioned where the catalytic water is expected for peptidic substrates, a partial negative charge at the hydroxyl O2 is stabilized by the adjacent Glu198, thereby strengthening its binding to Zn2+ (Figs. 2 and 3, TableII). The enol form of the barbiturate is thus favored by the protein matrix over the tautomeric keto form, which dominates in solution (37Budavari S. ONeil M.J. Smith A. Heckelman P. E The Merck Index.11th Ed. Merck % Co., Inc., Rahway,NJ1989Google Scholar). In addition, the polar H1-N1-C6=O6 atoms of the barbiturate (Fig. 3 B) mimic the P1′-S1′ antiparallel main chain interactions of a substrate (Fig. 3 A). The amide N1-H1 thereby is hydrogen bonded to the carbonyl of Ala161, and the ketone C6=O6 is stabilized by the amides of Leu160 and Ala161. This latter interaction is reminiscent of the oxyanion hole binding of serine proteinases, although here there is no evidence of oxygen anion stabilization. The C4=O4 ketone seems unlikely to contribute to the binding energy for two reasons. First, unfavorable geometry (TableI) precludes a role as a third ligand in the Zn2+ chelation. More importantly, the C4=O4 ketone would collide with the carbonyl oxygen position of Pro217 at the "southern" rim of the active site as defined by other MMP-8 structures (2Grams F. Reinemer P. Powers J.C. Kleine T. Pieper M. Tschesche H. Huber R. Bode W. Eur. J. Biochem. 1995; 228: 830-841Crossref PubMed Scopus (178) Google Scholar, 14Bode W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (323) Google Scholar, 26Brandstetter H. Engh R.A. von Roedern E.G. Moroder L. Huber R. Bode W. Grams F. Protein Sci. 1998; 7: 1303-1309Crossref PubMed Scopus (47) Google Scholar). Instead, the inhibitor induces a reorientation at the Pro217position at an energy cost we discuss below.Figure 2The RO200-1770 inhibitor bound to the active site of MMP-8 (yellow). The 2Fo − Fc electron density map is contoured 1.0 ς over the mean. Pro217(red) from a reference structure was superimposed to the protein model (yellow) to illustrate the conflict of its carbonyl oxygen with barbiturate binding. The figure was prepared by using the program MAIN (34Turk D. Chemistry. Technische Universität München, Munich, Germany1992Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIProfiling of MMP inhibitors (IC50 [nm])InhibitorMMP-8MMP-2MMP-9MT1-MMPMMP-1MMP-3TACERO200-1770170028702000670513030000ND2-aND, not determined.RO204-1924319039403160205025,00030000NDRO206-002725021041083027003400NDI-COL 04326034481135022,0003500>27,000RO206-0032482021621014,0002700>22,0002-a ND, not determined. Open table in a new tab Table IZn2+ coordination geometry: bonds, angles, and dihedral anglesZn2+–N3Bar2.09 ÅZn2+–O2Bar2.93 ÅZn2+–O4Bar3.15 ÅZn2+Nɛ21972.03 ÅZn2+Nɛ22011.95 ÅZn2+Nɛ22072.00 ÅZn2+O2Bar C2Bar78.2°Zn2+O4Bar C4Bar70.4°Trigonal bipyramidal description of the Zn2+ coordinationZn2+ Nɛ2197 Nɛ2207O2Bar−4.8°O2BarZn2+ Nɛ2197101.5°Nɛ2197 Zn2+Nɛ2207119.7°Nɛ2207Zn2+ O2Bar137.8°Nɛ2201 Zn2+N3Bar125.6°Square pyramidal description of the Zn2+ coordinationNɛ2207O2Bar N3BarNɛ220115.2°Nɛ2197Zn2+ Nɛ2207119.7°Nɛ2197 Zn2+N3Bar95.5°Nɛ2197Zn2+ O2Bar101.5°Nɛ2197 Zn2+Nɛ2201100.3°Pentagonal pyramidal description of the Zn2+ coordinationNɛ2207 O2BarO4Bar Nɛ220120.0°Nɛ2197 Zn2+O4Bar88.0° Open table in a new tab The pentacoordinated Zn2+ binding geometry resembles a highly distorted trigonal bipyramidal structure with O2, Nε2(His197), and Nε2(His207) approximately in plane with the Zn2+ ion, with Nε2(His201) and N3lying above and below the basal plane, respectively (Table I). Alternatively, the coordination can be described as a distorted square pyramid where O2, N3, Nε2(His201), and Nε2(His207) form the basal ligands (dihedral deviation from planarity 15°, Table I). The metal ion lies outside of the basal plane but within 0.5 Å, and the fifth ligand Nε2(His197) forms the apex of the pyramid Table I. (If considering O4 to be a sixth ligand, the geometry may be described as a pentagonal pyramid with O4 as basal ligand in addition to O2, N3, Nε2(His201), and Nε2(His207)). In contrast to the polar interactions of the barbiturate ring, the interactions mediated by the phenyl and piperidyl rings are predominantly hydrophobic and involve the S1′ and S2′ pockets, respectively. The most prominent interaction in the S1′ pocket is the ideally parallel planar stacking of the phenyl ring and His197 at a distance of 3.6 Å (Fig.4). The conserved Leu160contributes to ligand binding also with its side chain in the S1′ site. The phenyl ring does not by itself fill the S1′ site, but leaves space filled by a network of three ordered water molecules. The first of these (Sol595) is probably incompletely occupied and forms hydrogen bonds with the inhibitor, with MMP-8, and with a second water molecule. The proximity of the inhibitor phenyl fC4 atom to Sol595 (3.1 Å) indicates a O … H-C interaction (38Wahl M.C. Sundaralingam M. Trends Biochem. Sci. 1997; 22: 97-102Abstract Full Text PDF PubMed Scopus (326) Google Scholar). The carbonyl group of Leu193 forms a 2.9-Å hydrogen bond with Sol595 with, however, an unfavorable C=O193-O595 angle of 113°. The second water molecule, Sol602, is positioned deeper inside the S1′ pocket at a hydrogen bonding distance of 2.7 Å from Sol595. Sol602 in turn is hydrogen-bonded (2.8 Å) with the third solvent molecule in the S1′ pocket, Sol592. Sol592 is in a channel bounded by Arg222, which forms a link between the three water network in S1′ and, via Sol667 (2.9 Å from Sol602), water in the adjacent cavity. Mutation of Arg222 would connect the two cavities, opening a "back door" to S1′ for solvent access. The guanidinium group atoms Nη2 and Nη1 of Arg222 are fixed by hydrogen bonds to the carbonyl oxygen of Ala213 (3.3 Å) and the Oη of Tyr227 (3.3 Å), respectively. Since most MMPs lack an equivalently stabilized Arg, MMP-8 has a comparatively restricted S1′ site. The hydrophobic interactions of the piperidine ring are mediated by aliphatic surfaces from Pro217-Asn218-Tyr219 at the "southern" rim of S2′ and by the main chain Gly158-Ile159-Leu160 at the "northern" rim. The latter residue (Leu160) separates the S1′ and S2′ pockets. No ordered water molecule can be detected in the vicinity of the hydroxyl group pOH9, although the position of this solvent exposed ethanol group is well defined by electron density (Fig. 2) and is thus presumably hydrated by disordered water. Significant differences are apparent in the protein structure compared with previously determined MMP-8 structures (2Grams F. Reinemer P. Powers J.C. Kleine T. Pieper M. Tschesche H. Huber R. Bode W. Eur. J. Biochem. 1995; 228: 830-841Crossref PubMed Scopus (178) Google Scholar, 14Bode W. Reinemer P. Huber R. Kleine T. Schnierer S. Tschesche H. EMBO J. 1994; 13: 1263-1269Crossref PubMed Scopus (323) Google Scholar, 21Betz M. Huxley P. Davies S.J. Mushtaq Y. Pieper M. Tschesche H. Bode W. Gomis-Rüth F.X. Eur. J. Biochem. 1997; 247: 356-363Crossref PubMed Scopus (88) Google Scholar). The catalytic Zn2+ ion of the three reference structures occupies the same position to within 0.2 Å; it is, however, shifted from that average position by 0.6 Å in the RO200-1770 complex structure. Corresponding shifts of the Zn2+ protein ligand positions are also apparent, with the respective Nε2 and Cγ values measured as follows: His197 (0.4 Å, 0.3 Å), His201 (0.3 Å, 0.3 Å), and His207 (0.6 Å, 0.7 Å). Consistent with this overall shift, the side chain of the catalytic Glu198 is translated by 0.2 Å. This displacement of the catalytic Zn2+ and its protein ligands is evidently induced by inhibitor binding, as the net effect of the optimization of barbiturate-Zn chelation geometry and the inhibitor orienting forces arising from the other inhibitor-protein interactions. Of the two partial sequences harboring the Zn2+ binding histidine residues, the loop Ala206-His207-Asn218 is more exposed to the solvent and anchored by fewer protein contacts than the internal helix L191–H197EXXH201–L203. The conformation of this loop is altered by several effects associated with the binding of RO200-1770. First, the greater inherent plasticity of this loop leads to greater compensation by the Zn ligand His207 for shear stresses induced at the catalytic site. Second, the Pro217–Asn218 peptide bond is rotated by ∼100°, evidently to prevent a repulsive interaction between the barbiturate C4=O4 keto group with the Pro217 carbonyl. Third, residues Ser209, Tyr216, Pro217, and Asn218 form crystal contacts. These effects in combination lead to a translation along the entire loop from Ala206 to Pro217, which, however, is relatively rigid, leaving most dihedral angles similar to those in the reference structures. In the "north" rim of the active site, the largest change compared with the inhibitor free MMP-8 structure is a 0.98 Å displacement and disorder of the Ile159 side chain; the electron density shows a branched but symmetric side chain interpretable as two equally populated rotamers, which "swap" Cγ1 and Cγ2 positions. Utilizing the crystal structure of the MMP8-RO200-1770 complex, several follow-up compounds were synthesized and tested against the panel of metalloenzymes shown in Table II. The lead compound RO200-1770 shows broadly nonspecific micromolar inhibition, excepting only stromelysin 1 (MMP-3) with its ∼10-fold weaker binding affinity to RO200-1770. To facilitate synthesis, the piperidine of the lead compound RO200-1770 was substituted by an essentially isosteric piperazine, RO204-1924. The almost uniform decrease in binding affinity might be rationalized by higher desolvation penalties for piperazine binding. The theoretical clogP values calculated for 1,4-dimethylpiperidine (1.9) and 1,4-dimethylpiperazine (0.8) support this hypothesis (39Ghose A.K. Viswanadhan V.N. Wendoloski J.J. J. Phys. Chem. 1998; 102: 3762-3772Crossref Scopus (629) Google Scholar). I-COL043 and RO206-0027 represent the results of two orthogonal approaches to optimize P1′-S1′ and P2′-S2′ binding, respectively. For each inhibitor, an ∼10-fold increase in inhibition toward MMP-8, -2, -9, and -3 was accomplished, while inhibition of MT1-MMP and MMP-1 was weakened or remained relatively unchanged. With its 4-fold weaker inhibition of MMP-1, I-COL043 showed significantly enhanced selectivity potential against the latter enzyme. The P1′ and P2′ optimizations of I-COL043 and RO206-0027 are combined in RO206-0032 and the inhibition values demonstrate, to a first approximation, additivity of the effects for MMP-8, -2, -9, and MT1-MMP. The improvement in its binding affinity to stromelysin (MMP-3) is less distinct, while fibroblast collagenase (MMP-1) binding averages rather than