Structure of the Catalytic Domain of Fibroblast Collagenase Complexed with an Inhibitor

Abstract

Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor

“…Because Asp 153 is in NOE contact with His 151 and is fully conserved, it is the best candidate to provide the fourth ligand to complete an ordinary tetrahedral coordination geometry. The recent X-ray structures have confirmed these observation, and the metal-ligand distances reported by Lovejoy et al (1994a) have been included in Fig. 7.…”

“…At the first calcium site in the loop between strands 111 and IV, NOES from the backbone of conserved Gly 159, conserved Gly 161, and Val 163 to either of the calcium-binding Asp 158 or Glu 184 residues are consistent with the crystallographic observation of three additional calcium-binding carbonyl groups. These are the carbonyl oxygens of the equivalent two glycines and asparagine of fibroblast collagenase (MMP-1; Lovejoy et al, 1994a). Because the six distances of calcium to ligand at this site, ranging from 1.8 to 2.5 A, reported by Lovejoy et al (1994a) are consistent with the NOE data, they have been included in this model.…”

“…These are the carbonyl oxygens of the equivalent two glycines and asparagine of fibroblast collagenase (MMP-1; Lovejoy et al, 1994a). Because the six distances of calcium to ligand at this site, ranging from 1.8 to 2.5 A, reported by Lovejoy et al (1994a) are consistent with the NOE data, they have been included in this model. Some crystallographic models of collagenase include a third calcium ion just beyond the opposite edge of the sheet between the loops feeding into the N-terminal ends of @-strands Ill and V. A structural zinc also stabilizes the @-sheet.…”

“…Reported locations include: in the active site of a neighboring protease molecule ( Lovejoy et al,3994b); in its own active site (Gooley et al, 1994); pointing away into "solution" (i.e., the crystal) Borkakoti et al, 1994;Stams et al, 1994); and along thecarboxyterminal helix C (Lovejoy et al, 1994a;Reinemer et al, 1994). Like the latter two reports, the mature N-terminus (Phe 83) of the catalytic domain of stromelysin in this NMR model runs along the surface of helix C. Reinemer et al (1994) reported that the amino group of the N-terminal phenylalanine forms a salt bridge with the fully conserved Asp 232 in neutrophil collagenase properly processed at the correct N-terminal residue to give full activity.…”

“…These structural features, shared not only among the matrix metalloproteinases and astacins, but also among the serralysins and adamalysins, allow them to be grouped into a superfamily dubbed the metzincins (Bode et al, 1993). In efforts to aid drug design, crystallographic structures of the catalytic domain of fibroblast collagenase or MMP-1 (Borkakoti et al, 1994;Lovejoy et al, 1994aLovejoy et al, , 1994b and of neutrophil collagenase or MMP-8 Reinemer et al, 1994;Stams et al, 1994) have become available. The structure of full-length porcine MMP-I shows that the C-terminal hemopexin-like domain is a four-bladed ''0 propeller" (Li et al, 1995).…”