The Effect of Matrix Metalloproteinase Complex Formation on the Conformational Mobility of Tissue Inhibitor of Metalloproteinases-2 (TIMP-2)

Abstract

The backbone mobility of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) was determined both for the free protein and when bound to the catalytic domain of matrix metalloproteinase-3 (N-MMP-3). Regions of the protein with internal motion were identified by comparison of the T 1 and T 2 relaxation times and 1 H-15 N nuclear Overhauser effect values for the backbone amide 15 N signals for each residue in the sequence. This analysis revealed rapid internal motion on the picosecond to nanosecond time scale for several regions of free N-TIMP-2, including the extended ␤-hairpin between ␤-strands A and B, which forms part of the MMP binding site. Evidence of relatively slow motion indicative of exchange between two or more local conformations on a microsecond to millisecond time scale was also found in the free protein, including two other regions of the MMP binding site (the CD and EF loops). On formation of a tight N-TIMP-2⅐N-MMP-3 complex, the rapid internal motion of the AB ␤-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB ␤-hairpin is not a feature of all members of the TIMP family; therefore, the binding of this highly mobile region to a site distant from the catalytic cleft of the MMPs suggests a key role in TIMP-2 binding specificity.Breakdown of the extracellular matrix is an important event in many normal and pathological processes, such as growth, wound repair, tumor metastasis, and arthritis (1-3). A large family of zinc-dependent proteinases, the matrix metalloproteinases (MMPs), 1 are thought to be primarily responsible for this matrix catabolism. The activities of the MMP family in the extracellular matrix are highly regulated by transcriptional control, zymogen activation, and inhibition by a family of specific protein inhibitors, the tissue inhibitors of metalloproteinases or TIMPs (4). The TIMPs bind tightly to the active proteinases to form an inactive TIMP⅐MMP complex (5). Four mammalian TIMP proteins have now been identified (TIMP-1 to -4) (6 -9), and their high degree of sequence similarity and conservation of 12 Cys residues suggests that each consists of the same basic fold but with some variations in loop structures and glycosylation. The location of the MMP inhibitory site on the TIMP molecule has been shown to reside predominantly in the N-terminal two-thirds of the protein, defined by the first three disulfide bonds. This domain (N-TIMP) can be expressed independently to generate a fully folded, stable, and active inhibitor (10, 11).High resolution three-dimensional structures are now available for both the active N-terminal domain of TIMP-2 (12) and for the full-length inhibitor (13). Crystal structures have also been published for full-length TIMP-1 and TIMP-2 in complexes with the catalytic domains of MMP-3 (N-MMP-3) and MT1-MMP, respectively (14, 15). The structures of the TIMP⅐MMP complexes, together with NMR data on chemical shift perturbation seen for N-TIMP-2 on complex fo...

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

“…This finding further supports our previous suggestion that although the position of the extended AB hairpin will necessitate its close contact with a proteinase bound at the inhibitory site of TIMP-2 (see Fig. 1), this interaction need not contribute to the overall binding affinity in all cases and could, in some cases, conceivably weaken the overall binding interaction by making unfavorable contacts with the proteinase (6). The crystallographic data for the TIMP-2⅐MMP-14 complex have shown that the side chain of Tyr-36 is positioned on the surface of the MMP-14 catalytic domain in a cavity formed by the MT loop, the side chains of Asp-212 and Phe-180, and the S loop, which allows the formation of a hydrogen bond between the tyrosine hydroxyl group and the carboxylate oxygens of Asp-212 as well as with a number of Van der Waals contacts (4).…”

Mycobacterium tuberculosis RNA Polymerase-binding Protein A (RbpA) and Its Interactions with Sigma Factors

“…Because it was not possible to assign the NMR signals for the C-terminal region of RbpA, we cannot map the precise residues involved. It is not unusual for conformational averaging caused by protein flexibility to occur at high affinity protein-protein interaction sites; indeed there are many documented examples (52,(61)(62)(63)(64). The N and C termini are on the same side of the structured core (Fig.…”

Flexibility and Variability of TIMP Binding: X-ray Structure of the Complex Between Collagenase-3/MMP-13 and TIMP-2

“…Earlier NMR studies on the interaction of N-TIMP-2 with cdMMP-3 indicated a key role of the sA-sB loop of TIMP-2 for binding specificity. 22 It is thus clear that the small sV-hB loop of MMP-13 allows a closer approach of the Cterminal subdomain towards the enzyme than the more exposed loop of MT1-MMP, and that the bulkier S-loop of MMP-13 enforces a deviation of the C-connector loop. Both contacts are in agreement with the overall tilt observed in the two TIMP-2 complexes.…”