Characterization of the AB Loop Region of TIMP-2

Abstract

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is unique as it is the only member of the TIMP family that is involved in the cellular activation of promatrix metalloproteinase-2 (pro-MMP-2) by virtue of forming a trimolecular complex with membrane type 1 matrix metalloproteinase (MT1-MMP) on the cell surface. TIMP-4 is similar in structure to TIMP-2 but is unable to support the activation of the proenzyme. Several reports have highlighted the importance of the TIMP-2 C-terminal domain in the pro-MMP-2 activation complex; however, very little is known about the role of the extended AB loop of TIMP-2 in this mechanism even though it has been shown to interact with MT1-MMP. In this study we show by mutagenesis and kinetic analysis that it is possible to transfer the MT1-MMP binding affinity of the TIMP-2 AB loop to TIMP-4 but that its transplantation into TIMP-4 does not endow the inhibitor with pro-MMP-2 activating activity. However, transfer of both the AB loop and C-terminal domain of TIMP-2 to TIMP-4 generates a mutant that can activate pro-MMP-2 and so demonstrates that both these regions of TIMP-2 are important for the activation process. Matrix metalloproteinases (MMPs)2 have important and diverse roles in the remodeling of the extracellular matrix during development and disease (reviewed in Refs. 1-3). MMP activity is associated with tumor growth, metastasis, and neovascularization. The activities of various MMPs are controlled at the protein level via inhibition and activation by the following four homologous tissue inhibitors of metalloproteinases (TIMPs): TIMP-1, TIMP-2, TIMP-3, and TIMP-4, the most recent member of the family. TIMPs inhibit the MMPs in a 1:1 stoichiometry by tightly binding to the active site of the enzyme primarily through their N-terminal domain (4). However, TIMP binding and inhibition are improved by contacts with sites on the C-terminal domain of both the enzyme and the inhibitor (5). All four TIMPs inhibit active forms of most MMPs, but some differences in their inhibitory properties have been reported. For example, TIMP-2, TIMP-3, and TIMP-4 are effective inhibitors of the membrane-bound MMPs, whereas TIMP-1 is a very poor inhibitor of these enzymes (6). TIMPs have no inhibitory activity against astacins, but TIMP-3 and to some extent TIMP-1 have been shown to be active against the ADAMs. TIMP-3 inhibits ADAM-10, -12, and -17 and the aggrecan-degrading enzymes ADAM-TS4 and ADAM-TS5, whereas TIMP-1 inhibits ADAM-10 (7-9). The association of TIMPs with pro-MMP-2 and pro-MMP-9 is also specific. TIMP-2 binds to pro-MMP-2 through the hemopexin-like domain of the enzyme and facilitates enzyme activation, an action that is unique to TIMP-2; TIMP-1 binds to pro-MMP-9, whereas TIMP-3 binds to both proenzymes (10 -12). TIMP-4 also binds to pro-MMP-2 through the hemopexin-like domain (13); however, it is unable to promote the activation of the zymogen by MT1-MMP (14, 24, 28), although it is an excellent inhibitor of the latter. Recent mutagenesis studies by several groups have identified...

Tissue Inhibitor of Metalloproteinases-4. The road less traveled

“…Crystallographic, NMR and site-directed mutagenesis studies have revealed that the AB-loop is variably involved in the contacts between TIMPs and MMPs [ 14 - 16 , 23 ]. It has been shown that deleting this loop dramatically decreases the rate of association and increases the equilibrium constant of TIMP-2 binding to the catalytic domain of MT1-MMP (MMP-14) [ 33 ]. It was demonstrated that Tyr36, which is unique within the TIMP-2 sequence and located at the tip of the AB-loop, critically contributes to the interactions with MT1-MMP [ 34 ].…”

“…It was demonstrated that Tyr36, which is unique within the TIMP-2 sequence and located at the tip of the AB-loop, critically contributes to the interactions with MT1-MMP [ 34 ]. Notably, the deletion of the AB-loop in TIMP-4 exerts a considerably smaller effect in the kinetics and thermodynamic of binding with MT1-MMP, suggesting a different contribution of the TIMP-4 AB-loop for the MT1-MMP binding [ 33 ]. TIMP-4, which associates with the catalytic domain of MT1-MMP at a 20-fold slower rate than TIMP-2, has Ala instead of Tyr at position 36.…”

Timp‐2 binding with cellular MT1‐MMP stimulates invasion‐promoting MEK/ERK signaling in cancer cells

“…13,14 This binding mode is essential for the cell surface activation of proMMP-2 by MT1-MMP. 4,15 The peptide sequence of the PEX domain is conserved in the MMP family. This domain regulates the interactions of MT1-MMP with its cleavage targets including CD44 and the a integrins.…”

<p>MMP-2, MMP-9, TIMP-1, and TIMP-2 in the Peripheral Blood of Patients with Differentiated Thyroid Carcinoma</p>

“…26 TIMP-2 prevents self-activation of MMP-2 mainly by forming stable complexes with the MMP-2 precursor, or directly inhibits the catalytic activity of MMP-2 by combining with it. 27 TIMP-1 is a specific inhibitor of MMP-9. Previous studies 28 found that TIMP-1 expression could be detected in fibroblasts of DTC and thyroid adenoma, and it is closely related with tumor size, lymph node metastasis, clinical stage, and vascular invasion, while promoting recurrence.…”