TI  - The catalytic domain of activated <prot>collagenase I</prot> ( <prot>MMP-1</prot> ) is absolutely
      required for interaction with its specific inhibitor, <prot>tissue inhibitor of
      metalloproteinases-1</prot> ( <prot>TIMP-1</prot> ).
PG  - 81-8
AB  - Here, we describe the production of <prot>recombinant human tissue inhibitor of
      metalloproteinases-1</prot> (<prot>rTIMP-1</prot>) and wild-type and mutant human <prot>collagenase
      type I</prot> (<prot>rMMP-1</prot>) proteins in SF9 cells by the baculovirus expression
      system. Wild-type <prot>MMP-1</prot>, as well as the <prot>MMP-1</prot> mutant lacking the
      C-terminal hemopexin-like domain [des-(248-450)-<prot>MMP-1</prot>], exhibit enzymatic
      activity upon cleavage of the prodomain by treatment with <prot>trypsin</prot> or
      4-aminophenylmercuric acetate. Enzyme activity of both proteins can be
      inhibited by addition of <prot>rTIMP</prot>. Deletion of the complete active-site
      [des-(161-228)-<prot>MMP-1</prot>] within the catalytic domain, or mutation of a single
      His residue of the Zn2+ binding domain (His199), generates stable forms of
      <prot>MMP-1</prot> proteins which are unable to digest <prot>collagen type I</prot> or <prot>beta-casein</prot>.
      In addition to co-immunoprecipitation analysis, we have established a
      rapid and sensitive ELISA assay using immobilized <prot>rTIMP</prot> to determine the
      structural requirements of <prot>MMP-1</prot> to form complexes with its inhibitor.
      Only the activated and not the latent forms of wild-type and C-terminal
      mutant des-(248-450)- <prot>MMP-1</prot>  proteins are able to form complexes with  <prot>TIMP</prot> .
      Neither mutation of His199, nor deletion mutants des-(161-228)-<prot>MMP-1</prot> and
      des-(161-228/248-450)-<prot>MMP-1</prot>, interact with <prot>TIMP</prot>. This demonstrates that
      the C-terminal hemopexin domain of <prot>MMP-1</prot>, in contrast to the corresponding
      regions of <prot>gelatinase A</prot> and <prot>gelatinase B</prot>, does not interact with <prot>TIMP-1</prot>.
      In summary, we have shown that the integrity of the catalytic domain of
       <prot>MMP-1</prot>  and its ability to bind Zn2+ is absolutely required for complex
      formation with  <prot>TIMP-1</prot> , which further underlines the importance of this
      region for proper regulation of enzymatic activity of <prot>MMP-1</prot>.
AD  - Deutsches Krebsforschungszentrum Heidelberg, Abteilung Signaltransduktion
