The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: A kinetic analysis of the inhibition of gelatinase A

Abstract

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

The C‐terminal (haemopexin‐like) domain structure of human gelatinase A (MMP2): structural implications for its function

“…This is of importance to the inhibition of the active enzyme, because the interaction of the CTDs of TIMP-2 and MMP2 allows the N-terminal domain of TIMP-2 efficiently to dock with the enzyme's active site [22]. Sequence comparison of the TIMPs revealed that TIMP-2 possesses a negatively charged 'tail'.…”

“…Sequence comparison of the TIMPs revealed that TIMP-2 possesses a negatively charged 'tail'. Removal of this tail decreased the association rate constant of TIMP-2 and active MMP2 and prevented the inhibitor from binding to proMMP2 [22]. It is possible, therefore, that the positively charged region located on the exit side and the periphery of blade III of the MMP2-CTD represents an anchoring site for TIMP-2.…”

“…Indeed, the recent isolation of ternary complexes of proMMP2, TIMP-2, and MT-MMP (a membrane-bound MMP that activates proMMP2 [20]) suggests that the binding of TIMP-2 is of central importance to MMP2 function [21]. CTD interactions also assist in the inhibition of active MMP2 by TIMPs 1 and 2 [22].…”

Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP‐8) to stability and collagenolytic activity by alanine scanning mutagenesis

“…Collagenolytic activity was measured using 14 C-labelled fibrillar type I collagen at 35°C [25]. The concentrations of active wild-type and mutant neutrophil collagenase were determined by active site titrations using a standard TIMP-1 solution of known concentration and residual activity was monitored using the quenched fluorescence substrate as described above [26].…”

Specific, High Affinity Binding of Tissue Inhibitor of Metalloproteinases-4 (TIMP-4) to the COOH-terminal Hemopexin-like Domain of Human Gelatinase A

“…Gelatinase A is unique among the MMPs in that the latent enzyme binds TIMP-2 to form a tightly bound 1:1 molar stoichiometric complex (22,(33)(34)(35), whereas only the active forms of the other MMPs can bind this inhibitor. Binding of TIMP-2 to progelatinase A occurs via the C domains of the enzyme (17,18,34,36) and inhibitor (37,38); and with the other MMPs, an additional binding site for the N domain of TIMP-2 is also present in the catalytic domain of active gelatinase A (18,22,33,34). A further binding site for the N domain of TIMP-2 is also found on the C domain of gelatinase A (39), although the biological significance of this interaction is not yet clear.…”