The specificity of TIMP-2 for matrix metalloproteinases can be modified bysingle amino acid mutations

Residues 1-127 of human TIMP-8 (N-TIMP-2), comprising three of the disulfid e-bonded loops of the TIMP-8 molecule, is a discrete protein domain that fo lds independently of the C-terminal domain. This domain has been shown to b e necessary and sufficient for metalloproteinase inhibition and contains th e major sites of interaction with the catalytic N-terminal domain of active matrix metalloproteinases (MMPs), Residues identified as being involved in the interaction with MMPs by NMR chemical shift perturbation studies and T IMP/MMP crystal structures have been altered by site directed mutagenesis. We show, by measurement of association rates and apparent inhibition consta nts, that the specificity of these N-TIMP-2 mutants for a range of MMPs can be altered by single site mutations in either the TIMP "ridge" (Cys(1)-Cys (3) and Ser(68)-Cys(72)) Or the flexible AB loop (Ser(31)-Ile(41)). This wo rk demonstrates that it is possible to engineer TIMPs with altered specific ity and suggests that this form of protein engineering may be useful in the treatment of diseases such as arthritis and cancer where the selective inh ibition of key MMPs is desirable.