Journal of Molecular Biology
Volume 381, Issue 5, 19 September 2008, Pages 1307-1319
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Structural Determinants of the ADAM Inhibition by TIMP-3: Crystal Structure of the TACE-N-TIMP-3 Complex

https://doi.org/10.1016/j.jmb.2008.06.088Get rights and content

Abstract

TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-α converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the complex formed by the catalytic domain of recombinant human TACE and the N-terminal domain of TIMP-3 (N-TIMP-3), and determined its molecular structure with X-ray data to 2.3 Å resolution. The structure reveals that TIMP-3 exhibits a fold similar to those of TIMP-1 and TIMP-2, and interacts through its functional binding edge, which consists of the N-terminal segment and other loops, with the active-site cleft of TACE in a manner similar to that of matrix metalloproteinases (MMPs). Therefore, the mechanism of TIMP-3 binding toward TACE is not fundamentally different from that previously elucidated for the MMPs. The Phe34 phenyl side chain situated at the tip of the relatively short sA-sB loop of TIMP-3 extends into a unique hydrophobic groove of the TACE surface, and two Leu residues in the adjacent sC-connector and sE-sF loops are tightly packed in the interface allowing favourable interactions, in agreement with predictions obtained by systematic mutations by Gillian Murphy's group. The combination of favourable functional epitopes together with a considerable flexibility renders TIMP-3 an efficient TACE inhibitor. This structure might provide means to design more efficient TIMP inhibitors of TACE.

Introduction

Tumor necrosis factor-α (TNF-α), a major immunomodulatory and pro-inflammatory cytokine, is released from its membrane-anchored precursor via limited proteolysis by the TNF-α converting enzyme (TACE). TACE is a multidomain zinc-endopeptidase belonging to the “a disintegrin and metalloproteinase” (ADAM) family and the metzincin superfamily,1 which has been designated ADAM-17 (identified in the MEROPS database as M12.217 of subfamily B of family M12 of subclan MA(M) of clan MA).2 It is a type I membrane protein consisting of an N-terminal pro-domain, a metzincin-type catalytic domain, a disintegrin domain, two Cys-rich domains, a transmembrane helix and a cytoplasmic domain.3, 4, 5, 6 Besides its action on pro-TNF-α, TACE is believed to be involved in ectodomain shedding of several other cell surface-anchored bioactive molecules, such as the TNF receptors p75 and p55,7 and the transforming growth factor-α.8 Soluble forms of proteins shed by TACE are crucial for mammalian development, have a role in leukocyte migration, and participate in tumor cell proliferation as ligands of the epidermal growth factor receptor. Due to its involvement in many physiological processes and inflammatory diseases, regulation of the proteolytic activity of TACE is of utmost importance in the maintenance of the general well-being of the entire physiological system.9, 10

The four mammalian tissue inhibitors of matrix metalloproteinases (TIMP-1 to TIMP-4) were identified originally as powerful inhibitors of the matrix metalloproteinases (MMPs), blocking them non-covalently with 1:1 stoichiometry without much discrimination. However, later it was found that these TIMPs exhibit slightly different inhibition profiles, e.g. TIMP-1 interacts more weakly than TIMP-2 with the membrane-type MMPs and MMP-19.11, 12 In contrast to the other TIMPs, TIMP-3 (identified in the MEROPS database as I35.033 of family I35 of clan IT) is expressed during some specific cellular events in relatively small amounts,13 and is further unique in that it interacts strongly with extracellular matrix (ECM) components.14 TIMP-3 is known for its ability to induce apoptosis in mammalian cells,15 while mutations in its C-terminal domain cause the rare hereditary blindness Sorsby's fundus dystrophy.16

On the basis of the structure of the MMP-3*TIMP-1 complex,17 and due to the presence of similar TIMP interaction sites in TACE, we concluded that, in principle, the TIMPs should be able to interact with ADAMs such as TACE.18 Eventually, TACE was found to be inhibited efficiently by TIMP-3, but is blocked only very weakly by the other three mammalian TIMPs.19, 20 Interestingly, the inhibitory power of these active TIMP variants differs towards the catalytic domain (cdTACE) and the long soluble multidomain TACE form (ectoTACE).21, 22 Furthermore, TIMP-3 is able to selectively inhibit some other adamalysins, such as ADAM-1023 and ADAM-12-S,24 besides some ADAMs with thrombospondin motifs (ADAMTSs), such as ADAMTS-4 and ADAMTS-5,25, 26 and ADAMTS-2.27 TIMP-1 and TIMP-2, in contrast, seem to exhibit some inhibitory power only against a few distinct ADAMs; namely, ADAM-1023 and ADAM-12,28 respectively. In fact, the spectrum of metzincin targets of TIMP-3 is unmatched by that of any other TIMP.29

Gillian Murphy and her group have prepared a variety of TIMP-1 and TIMP-2 mutants and systematically replaced some surface epitopes to identify critical structural elements in the TIMPs responsible for this selectivity.29, 30 Indeed, they have been able to create a TIMP-2 variant with an inhibitory potency to TACE almost equal to that of TIMP-3,29 and to transform N-TIMP-1 and N-TIMP-4 (representing the inhibitory active N-terminal fragments of TIMP-1 and TIMP-4) into potent TACE inhibitors by transplanting some epitopes presumed to be beneficial for TACE binding.31, 32 TACE has been reported to be inhibited by TIMP-3 variants, in which the N-terminal α-amino group of Cys1 is modified chemically or substituted by an additional amino acid residue.33 This observation is in stark contrast to the MMPs, which do not form complexes with such N-terminally substituted TIMP species,34, 35, 36 in agreement with the tight packing of Cys1 in the interface of these MMP–TIMP complexes.17, 37, 38 Also, replacement of Thr2 by other residues decreased the binding affinity of N-TIMP-3 to TACE only modestly,39 again in contrast to the MMPs, which show great variations in affinity towards Thr2 mutants.40, 41 The N-TIMP-3 inhibition of TACE has been reported to display positive co-operativity, which was explained by the presence of multiple interacting binding sites and alternative conformational states.33 Taken together, these features of TIMP-3 suggest that the mechanism of TIMP-3 inhibition of TACE might be distinct from that of the MMPs.33

Interaction models constructed on the basis of the experimental crystal structures of the complexes between the catalytic domains of MMP-3 and MMP-14 with TIMP-117, 37 and TIMP-244 were not sufficient to explain these properties. In order to further delineate the molecular basis that governs the TIMP*TACE selectivity,29 we crystallized the complex of the catalytic domain of TACE (cdTACE) with human N-TIMP-3, which inhibits cdTACE with an affinity similar to that of full-length TIMP-3.42 The structural analysis of this cdTACE*N-TIMP-3 complex reveals that the TIMP inhibition mechanism towards the ADAMs is similar to, if not identical with, that of the MMPs, but that small differences of TIMP-3 seem to enable a more favourable interaction with TACE.

Section snippets

Overall structure

The cdTACE*N-TIMP-3 complex exhibits the shape of a compact, almost spherical mushroom, with the wedge-shaped N-TIMP-3 molecule constituting the expanded foot, and the cdTACE enzyme representing the half-spherical, slightly oblate cap (Fig. 1). The N-TIMP-3 inhibitor slots with its small edge into the entire active-site cleft of the catalytic domain of TACE, thereby burying solvent-accessible surfaces of 2120 Å2 (Fig. 1). Compared with the cdMMP-13*TIMP-238 and the cdMMP-3*TIMP-117 complexes,

Conclusion

The crystal structure of the cdTACE*N-TIMP-3 complex revealed that TIMP-3 exhibits a fold similar to the other TIMPs, and that it interacts with cdTACE in a manner virtually identical with that of TIMPs-1 and-2 with the MMPs (see Figs. 3a and b). However, in some substantial details N-TIMP-3 differs from models built on the basis of TIMPs-1 and 2. The cdTACE*N-TIMP-3 structure is in accordance with the rules underpinning TACE*TIMP-3 recognition as discovered by Gillian Murphy and her group:(1)

Proteins

The expression and purification of the human TACE catalytic domain have been described.18 Briefly, a construct of human TACE (1E-477E) containing the mutations Ser266EAla and Asn452EGln and a C-terminal Gly-Ser-(His)6 sequence was expressed in CHO cells, which secreted two forms of activated cdTACE starting with Val212E as well as with Arg215E. This mature proteinase mixture was purified by NTA-Ni affinity chromatography and by gel-filtration chromatography.

Human N-TIMP-3 (Cys1-Asn121) was

Acknowledgements

We are grateful to R. Faessler for his generous support of this work. The financial support by the European Commission (projects CAMP: LSHG-2006-018830 and CANCERDEGRADOME: LSH-2002-2.2) is gratefully acknowledged.

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