Matrilysin-inhibitor complexes: common themes among metalloproteases

Molecular Structure Department, Syntex Discovery Research, Palo Alto, California 94303, USA.
Biochemistry (Impact Factor: 3.02). 06/1995; 34(20):6602-10.
Source: PubMed


Matrix metalloproteases are a family of enzymes that play critical roles in the physiological and pathological degradation of the extracellular matrix. These enzymes may be important therapeutic targets for the treatment of various diseases where tissue degradation is part of the pathology, such as cancer and arthritis. Matrilysin is the smallest member of this family of enzymes, all of which require zinc for catalytic activity. The first X-ray crystal structures of human matrilysin are presented. Inhibitors of metalloproteases are often characterized by the chemical group that interacts with the active site zinc of the protein. The structures of matrilysin complexed with hydroxamate (maximum resolution 1.9 A), carboxylate (maximum resolution 2.4 A), and sulfodiimine (maximum resolution 2.3 A) inhibitors are presented here and provide detailed information about how each functional group interacts with the catalytic zinc. Only the zinc-coordination group is variable in this series of inhibitors. Examination of these inhibitor-matrilysin complexes emphasizes the dominant role the zinc-coordinating group plays in determining the relative potencies of the inhibitors. The structures of these matrilysin-inhibitor complexes also provide a basis for comparing the catalytic mechanism of MMPs and other metalloproteins.

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    • "BB94 is selected for positive binding and phosphoramidon for negative control on MMP7. The 3D structures of the human MMP7 protease [3] (PDB code: 1MMR) and thermolysin [10] (EC#: ((PDB code: 1THL) were retrieved from the Protein Data Bank. PRODRG program ( programs/prodrg/) were used to generate the coordinate and topology files of SC44463 [11,12], BB94 [13-15] and Phosphoramidon [16,17]. "
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    ABSTRACT: The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?" We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3 day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional "domain" found to date for the matrixin family. The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.
    Journal of Biomedical Science 05/2012; 19(1):54. DOI:10.1186/1423-0127-19-54 · 2.76 Impact Factor
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    • "This induced fit mechanism fails for MMP-7 due to its shorter specificity loop creating a more rigid S1 0 pocket. The shallow specificity pocket of MMP-1 can accommodate a diphenylether backbone of 1 (Terp et al. 2002; Browner et al. 1995). "

    EXS 01/2012; 103:v-vi.
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    • "This induced fit mechanism fails for MMP-7 due to its shorter specificity loop creating a more rigid S1 0 pocket. The shallow specificity pocket of MMP-1 can accommodate a diphenylether backbone of 1 (Terp et al. 2002; Browner et al. 1995). "
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    ABSTRACT: Matrix metalloproteinases (MMPs) regulate a wide range of biological functions; hence, they have invited great attention for the studies on their structures and functions, and since their overactivation leads to several diseases, the design and discovery of their potent inhibitors have become the need of the day. Since there have been so far discovered 28 different types of human MMPs, the specificity of binding of inhibitors with each different MMP needs special attention. The chapter presents the X-ray crystallographic and NMR studies on three-dimensional structures of a number of MMPs to reveal their catalytic site, subsites, specificity of binding with substrate and inhibitors, and catalytic mechanism. In addition to catalytic site, MMPs possess some subsites designated by unprimed and primed S, e.g., S1, S2, S3 and S1', S2', S3'. Among these, the S1' pocket varies the most among the different MMPs varying in both the amino acid makeup and depth of the pocket (shallow, intermediate, and deep pocket MMPs). This, along with the flexibility in the structures of MMPs, could be of great help in the design and the development of selective MMP inhibitors (MMPIs). The determination of affinity of inhibitors and the cleavage position of peptide substrates is mainly based on P1'-S1' interaction (P1', the group in inhibitor or substrate binding to S1' pocket of the enzyme), and it is the main determinant for the affinity of inhibitors and the cleavage position of peptide substrates.
    EXS 01/2012; 103:35-56. DOI:10.1007/978-3-0348-0364-9_2
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