Matrix metalloproteinases (MMPs) are zinc endopeptidases that are required for the degradation of extracellular matrix components during normal embryo development, morphogenesis and tissue remodelling 1. Their proteolytic activities are precisely regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs) 1, 2, 3, 4, 5. Disruption of this balance results in diseases such as arthritis, atherosclerosis, tumour growth and metastasis 1, 2. Here we report the crystal structure of an MMP-TIMP complex formed between the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1. TIMP-1, a 184-residue protein 5, has the shape of an elongated, contiguous wedge. With its long edge, consisting of five different chain regions, it occupies the entire length of theactive-site cleft of MMP-3. The central disulphide-linked segments Cys 1-Thr 2-Cys 3-Val 4 and Ser 68-Val 69 bind to either side of the catalytic zinc. Cys 1 bidentally coordinates this zinc, and the Thr-2 side chain extends into the large specificity pocket of MMP-3. This unusual architecture of the interface between MMP-3 and TIMP-1 suggests new possibilities for designing TIMP variants and synthetic MMP inhibitors with potential therapeutic applications.

The TIMP family at present comprises four members (TIMP-1 to TIMP-4) of relative molecular mass (M r) ranging from 22K to 30K, with 40–50% sequence identity 3. All TIMPs inhibit active MMPswith relatively low selectivity, forming tight non-covalent 1: 1 complexes 4. Native TIMP-1 contains two glycosylation sites 5. AC-terminally truncated form of TIMP-1 comprising only the N-terminal two-thirds of its polypeptide chain (N-TIMP-1) has been shown to inhibit MMP-3 with a slightly reduced affinity compared with full-length TIMP-1 (refs 4, 6, 7). These experiments suggested that TIMPs interact with MMPs predominantly through their N-terminal moiety. A preliminary nuclear magnetic resonance (NMR) model of N-TIMP-2 has been reported 8, but the mechanism of MMP inhibition by the TIMPs is not understood. Most MMPs, including stromelysin-1, are secreted as multimodular proenzymes that can be activated, with a 165-residue catalytic domain flanked by an N-terminal activation peptide and a spacer-linked C-terminal domain, respectively. Three-dimensional structures have been determined for a few catalytic MMP domains, including those of stromelysin-1 (MMP-3) 9, 10, 11 and its proform 10, and for full-length porcine MMP-1 (ref. 12). To understand the mechanism of interaction between MMPs and TIMPs, and to allow the design of morespecific TIMP species, we have undertaken an X-ray crystal-structure analysis of the complex formed between unglycosylated human TIMP-1 and the catalytic domain of MMP-3.