Tyrosine 36 Plays a Critical Role in the Interaction of the AB Loop of Tissue Inhibitor of Metalloproteinases-2 with Matrix Metalloproteinase-14

Abstract

The tissue inhibitor of metalloproteinases-2 (TIMP-2) is potentially an important inhibitor of all known matrix metalloproteinases (MMPs). However, it has been shown to undergo specific interactions with both MMP-2 (gelatinase A) and MMP-14 (MT1-MMP), and it has been proposed that these three proteins function as a cell surface-based activation cascade for matrix metalloproteinases and as a focus of proteolytic activity. In this study, we have carried out mutagenesis and kinetic analyses to examine the unique interactions between the AB loop of TIMP-2 and MMP-14. The results demonstrate that the major binding contribution of the AB loop is due solely to residue Tyr-36 at the tip of the hairpin. From this work, we propose that TIMP-2 may be engineered to abrogate MMP-14 binding, whereas its binding properties for other MMPs, including MMP-2, are maintained. Mutants of TIMP-2 with more directed specificity may be of use in gene therapeutic approaches to human disease.The role of the tissue inhibitors of metalloproteinases (TIMPs) 1 -1-4 in the regulation of matrix metalloproteinases (MMPs) and hence extracellular matrix protein turnover has now been well documented (reviewed in Refs. 1 and 2). One important focus of research has been the relationship of the structure of TIMPs to their function as MMP inhibitors, studies that have been significantly promoted by the determination of two crystal structures for MMP⅐TIMP complexes (3, 4) and NMR studies mapping the MMP binding site on N-TIMP-2 (5-7). Domain and site-directed mutagenesis studies on the TIMPs have highlighted both the basic similarities in their binding mechanisms to different MMPs and further unique interactions occurring between specific enzyme inhibitor pairs (8 -12). Our studies on TIMP-2 have demonstrated a specific biochemical and biological relationship with both MMP-2 and MMP-14, with the latter as part of a proposed cell surfacebased activation mechanism for MMPs (13). We have carried out kinetic studies on the interaction of N-TIMP-2 with various MMPs, showing that one such specific interaction could be demonstrated between the hairpin turn of the A and B ␤-strands of TIMP-2 (Tyr-36) and . This substantiated the structural study of the MMP-14⅐TIMP-2 complex (4), where the Tyr-36 at the tip of this loop was seen to fit into a cavity on the surface of the MMP-14 molecule, bordered by the MT loop and the side chains of Asp-212, Ser-189, and Phe-180. Fernandez-Catalan et al. (4) also described several other potentially significant interactions between residues of the TIMP-2 AB hairpin and MMP-14. We have therefore extended our mutagenesis and kinetic studies of this region (the tip of the AB hairpin) to establish the importance of these interactions for TIMP-2/MMP-14 binding. Site-directed residue changes were made at four sites in the AB hairpin of N-TIMP-2, and the entire tip of the hairpin was removed by the deletion of 7 residues (Asp-34 to Ile-40). The site of the residue substitutions in the three-dimensional structure of the TIMP-2⅐MMP-14 co...

Delineating the Molecular Basis of the Inactivity of Tissue Inhibitor of Metalloproteinase-2 against Tumor Necrosis Factor-α-converting Enzyme

“…Here, we showed that Phe 34 is the decisive element that sets the TIMP-3 AB-loop apart from those of TIMP-1, -2, and -4 when it comes to TACE selection. The only comparable example so far is the dissection carried out on the TIMP-2 AB-loop by Williamson et al (23). As shown in his report, mutation of the tyrosine 36 residue to glycine (Y36G) reduced the affinity of N-TIMP-2 for MT1-MMP by nearly a 100-fold (K i of 1.2 nM for wild-type N-TIMP-2 versus 124 nM for Y36G).…”

“…Situated at the edge of the MMP-binding ridge, the exact role of the AB-loop in MP selectivity has been a subject of constant speculation over the years. Indeed, the delineation of TIMP-2/MT1-MMP cocrystal structure (PDB 1BUV) in 1998 and subsequent mutagenesis study confirmed the significance of the AB-loop in MP association (23). In this study, two AB-loop mutants of the same length were created, each for a different reason.…”

Characterization of the AB Loop Region of TIMP-2

“…The structural authenticity of the constructs was confirmed by electrospray mass-spectrometry, and the constructs were found to be between 70 and 80% active by active site titration against MMP-2 (data not shown). Previous structural work has shown that the AB loop of TIMP is highly solvent-exposed and can accommodate changes to its sequence without any perturbation of the rest of the protein structure (19).…”

“…Design of AB Loop Mutants-In our previous kinetic study (19), we showed that the side chain of Tyr 36 in the AB loop of N-TIMP-2 was responsible for a specific interaction with the catalytic domain of MT1-MMP, increasing the rate of inhibition (k on ) by 180-fold and the affinity (K i ) by 110-fold when compared with the Y36G N-TIMP-2 mutant. N-TIMP-4 inhibits MT1-MMP cat with an affinity comparable with that of N-TIMP-2 but associates with the proteinase at a 20-fold slower rate (Table 1).…”

“…We had shown previously that Tyr 36 at the tip of the hairpin in TIMP-2 makes unique and specific interactions with MT1-MMP at a site away from the catalytic site of the enzyme (18,19). Our aim in this paper was to investigate the importance of this region in TIMP-4 for MT1-MMP inhibition and whether the extended AB loop region of TIMP-2 is required for the pro-MMP-2 activation complex.…”