Residue 2 of TIMP-1 is a major determinant of affinity and specificity formatrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1 ' residue of substrate

The unregulated activities of matrix metalloproteinases (MMPs) are implicat ed in disease processes including arthritis and tumor cell invasion and met astasis, NMP activities are controlled by four homologous endogenous protei n inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet differen t TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1.MMP-3 complex includes a contiguous region of TIMP- 1 around the disulfide bond between Cys(1) and Cys(70) that inserts into th e active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribut ion to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity o f residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP -1 interacts with the S1' specificity pocket of MMP-3, which is a key to su bstrate specificity, but the structural requirements in TIMP-1 residue 2 fo r MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substi tutions for Thr(2) represent suitable starting points for generating more t argeted TIMPs for investigation and for intervention in MMP-related disease s.