The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1⅐MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys 1 and Cys 70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of 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 substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr 2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases.The matrix metalloproteinases (MMPs) 1 are a family of about twenty Zn 2ϩ -dependent endopeptidases that have important roles in connective tissue turnover during physiological processes including development, morphogenesis, and wound healing (1, 2). Their activities in the extracellular matrix are stringently regulated through transcriptional control, zymogen activation, and the actions of four endogenous inhibitory proteins, tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 (3-7). Normal matrix homeostasis is associated with an appropriate balance between the levels of TIMPs and active MMPs, whereas an imbalance involving excess MMP activity is linked with disease processes including arthritis, tumor cell metastasis, and tissue invasion and atherosclerosis (1, 2).Mammalian TIMPs have an N-terminal domain of about 125 amino acids and a smaller C-terminal domain of about 65 amino acids; each domain is stabilized by three disulfide bonds (8). The N-terminal domains of different TIMPs fold into a correct native structure which carries the inhibitory activity against MMPs (9 -11). Although correctly folded and functional C-terminal domains have not been described, truncation experiments indicate that this region is responsible for the interactions of TIMPs with pro-MMPs (12, 13). There is little specificity in the inhibitory actions of TIMPs on metalloproteinases, with the exception of the ability of TIMP-2 and TIMP-3 to inhibit membrane-type metalloproteinases-1 and -2, whereas TIMP-1 is a poor inhibitor of these enzymes (12-14). However, the interactions of TIMPs with pro-MMPs are more specific. For example, TIMP-2 and TIMP-4 form specific complexes with pro-MMP-2 (progelatinase A), whe...

Differences in proteinase susceptibility between free TIMP-1 and the TIMP-1-MMP-3 complex and mutagenesis studies suggested that the residues around the disulfide bond between Cys1 and Cys70 in TIMP-1 may interact with MMPs. The crystal structure of the complex between TIMP-1 and the catalytic domain of MMP-3 has revealed that the alpha-amino group of Cys1 bidentately chelates the catalytic zinc of MMP-3 and the Thr2 side chain occupies the S1' pocket. Generation of the N-terminal domain of TIMP-1 (N-TIMP-1) variants with 15 different amino acid substitutions for Thr2 has indicated that the nature of the side chain of residue 2 has a major effect on the affinity of N-TIMP-1 for three different MMPs (MMPs-1, -2 and -3). The results also demonstrate that the mode of binding of N-TIMP-1 residue 2 differs from the binding of the P1' residue of a peptide substrate.

Aromatic cluster 1 of alpha-lactalbumin (LA), a substructure adjacent to the cleft, is important for its interaction with galactosyltransferase (GT) and effects on glucose binding in the lactose synthase complex [Grobler, J. A., Wang, M., Pike, A. K., & Brew, K. (1994) J. Biol. Chem. 269, 5106-5114]. The full extent of the functional region in LA has been probed by mutagenesis of residues that are near aromatic cluster 1 or within the cleft that corresponds to the active site in the homologous type c lysozymes. The conserved residues Val42, Gln54, and Ile59, which correspond to residues of lysozyme that act in substrate binding in subsites C to E, together with residues adjacent to aromatic cluster 1, were found to be not required for activity. In contrast, replacing Leu110, a component of the region corresponding to lysozyme subsite F, with His or Glu greatly reduces the affinity of LA for GT while the introduction of Arg lowers the synergism of LA and glucose binding to GT and also reduces the affinity of LA for GT. Substitutions for Ala106, which is adjacent to Leu110 in the structure, also perturb activity. The region of the cleft corresponding to subsite F is important for function in LA as well as in lysozyme since other components of this subsite, His32 and Phe31, are also crucial for LA activity. The qualitatively different effects of various substitutions for Leu110 may be mediated by their influence on His32 or by changes in the structure of the lactose synthase complex.

alpha-Lactalbumins and the type-c lysozymes are homologues with similar folds that differ in function and stability. To determine if the lower stability of alpha-lactalbumin results from specific substitutions required for its adaptation to a new function, the effects of lysozyme-based and other substitutions on thermal stability were determined. Unblocking the upper cleft in alpha-lactalbumin by replacing Tyr103 with Ala, perturbs stability and structure but Pro, which also generates an open cleft, is compatible with normal structure and activity. These effects appear to reflect alternative enthalpic and entropic forms of structural stabilization by Tyr and Pro. Of 23 mutations, only three, which involve substitutions for residues in flexible substructures adjacent to the functional site, increase stability. Two are lysozyme-based substitutions for Leu110, a component of a region with alternative helix and loop conformations, and one is Asn for Lys114, a residue whose microenvironment changes when alpha-lactalbumin interacts with its target enzyme. While all substitutions for Leu110 perturb activity, a Lys114 to Asn mutation increases T(m) by more than 10 degrees C and reduces activity, but two other destabilizing substitutions do not affect activity. It is proposed that increased stability and reduced activity in Lys114Asn result from reduced flexibility in the functional site of alpha-lactalbumin.

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