Mapping the Binding Site for Matrix Metalloproteinase on the N-Terminal Domain of the Tissue Inhibitor of Metalloproteinases-2 by NMR Chemical Shift Perturbation†

Tissue inhibitor of metalloproteinases (TIMP)-2 forms a noncovalent complex with the precursor of matrix metalloproteinase 2 (proMMP-2, progelatinase A) through interaction of the C-terminal domain of each molecule. We have isolated the proMMP-2-TIMP-2 complex from the medium of human uterine cervical fibroblasts and investigated the processes involved in its activation by 4-aminophenylmercuric acetate (APMA). The treatment of the complex with APMA-activated proMMP-2 by disrupting the Cys73-Zn2+ interaction of the zymogen. This is triggered by perturbation of the proMMP-2 molecule, but not by the reaction of the SH group of Cys73 with APMA. The ‘activated’ proMMP-2 (proMMP-2*) formed a new complex with TIMP-2 by binding to the N-terminal inhibitory domain of the inhibitor without processing the propeptide. Thus the APMA-treated proMMP-2*-TIMP-2 complex exhibited no gelatinolytic activity. In the presence of a small amount of free MMP-2, however, proMMP-2* in the complex was converted into the 65 kDa MMP-2 by proteolytic attack of MMP-2, but the complex did not exhibit gelatinolytic activity. The gelatinolytic activity detected after APMA treatment was solely derived from the activation of free proMMP-2. The removal of the propeptide of the proMMP-2* bound to TIMP-2 was also observed by MMP-3 (stromelysin 1), but not by MMP-1 (interstitial collagenase). MMP-3 cleaved the Asn80-Tyr81 bond of proMMP-2*. On the other hand, when MMP-3 was incubated with the proMMP-2-TIMP-2 complex, it bound to TIMP-2 and rendered proMMP-2 readily activatable by APMA. These results indicate that the blockage of TIMP-2 of the complex with an active MMP is essential for the activation of proMMP-2 when it is complexed with TIMP-2.

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Total Synthesis of Thielocin B1 and NMR Chemical Shift Perturbation Experiments with PAC3 Homodimer

Springer Theses - Total Synthesis of Thielocin B1 as a Protein-Protein Interaction Inhibitor of PAC3 Homodimer ◽

10.1007/978-4-431-55447-9_2 ◽

2015 ◽

pp. 25-62

Author(s):

Kosuke Ohsawa

Keyword(s):

Chemical Shift ◽

Total Synthesis ◽

Chemical Shift Perturbation ◽

Nmr Chemical Shift

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Synthesis of theC-terminal domain of the tissue inhibitor of metalloproteinases-1(TIMP-1)

Journal of Peptide Science ◽

10.1002/psc.455 ◽

2003 ◽

Vol 9 (7) ◽

pp. 430-441

Author(s):

József Bódi ◽

Nikolett Mihala ◽

Andrea Hajnal ◽

Katalin F. Medzihradszky ◽

Helga Süli-Vargha

Keyword(s):

Tissue Inhibitor Of Metalloproteinases ◽

Tissue Inhibitor ◽

Terminal Domain

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Differential functional significance of AP-1 binding sites in the promoter of the gene encoding mouse tissue inhibitor of metalloproteinases-3

Biochemical Journal ◽

10.1042/bj3240547 ◽

1997 ◽

Vol 324 (2) ◽

pp. 547-553 ◽

Cited By ~ 11

Author(s):

Hyungtae KIM ◽

William D. PENNIE ◽

Yi SUN ◽

Nancy H. COLBURN

Keyword(s):

Binding Site ◽

Binding Sites ◽

Functional Significance ◽

Regulatory Elements ◽

Binding Activity ◽

Model Systems ◽

Mouse Tissue ◽

Significant Activity ◽

Tissue Inhibitor Of Metalloproteinases ◽

Tissue Inhibitor

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular-matrix-associated protein that suppresses tumorigenicity or invasion in several model systems. We have identified, by in vitro footprinting, six AP-1 (activator protein-1) or AP-1-like binding sites in the mouse TIMP-3 promoter that bind purified c-Jun homodimers. Electrophoretic mobility shift assays revealed that the non-consensus fifth AP-1 binding site (AP-720; nt -720 to -714) had the strongest binding activity for recombinant c-Jun protein, and that the fourth binding site (AP-763; nt -763 to -754) and AP-720 showed strong binding activity for cellular nuclear proteins. Antibody supershift and blocking experiments suggest that AP-720, but not AP-763, binds authentic AP-1 components. Transient transfection reporter assays of deletion constructs showed that the region spanning AP-720 has the highest transcriptional activity, and that sequences 5′ to this region (nt -2846 to -747) may contain negative regulatory elements. The deletion construct containing about 500 nt 5′ to the transcriptional start, but no AP-1 sites, showed lower but significant activity, suggesting both AP-1-dependent and -independent regulation of the mouse TIMP-3 promoter. Mutational inactivation of AP-720 abolished the activity increment that distinguished the reporter construct containing both AP-720 and sixth AP-1 binding site (AP-617; nt -617 to -611) from that containing only AP-617. In summary, we report here that both AP-1 and non-AP-1 elements contribute to activity, with the non-consensus AP-1 site at -720 showing the greatest functional significance among the AP-1 sites.

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