Structural Determinants of the ADAM Inhibition by TIMP-3: Crystal Structure of the TACE-N-TIMP-3 Complex Magdalena Wisniewska 1 , Peter Goettig 1 , Klaus Maskos 1,2 , Edward Belouski 3 , Dwight Winters 3 , Randy Hecht 3 , Roy Black 4 and Wolfram Bode 1 ⁎ 1 Max-Planck-Institut für Biochemie, Proteinase Research Group, Am Klopferspitz 18, D-82152 Martinsried, Germany 2 Present address: Proteros Biostructures GmbH, Am Klopferspitz 19, D-82152 Martinsried, Germany 3 Amgen, Department of Protein Sciences,Thousand Oaks, CA, USA 4 Amgen, Department of Inflammation, 1201 Amgen Court West, Seattle, WA 98119-3105, USA Received 6 April 2008; received in revised form 20 June 2008; accepted 30 June 2008 Available online 7 July 2008 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. © 2008 Elsevier Ltd. All rights reserved. Edited by G. Schulz Keywords: TNF-α converting enzyme; a disintegrin and metalloproteinase; tissue inhibitor of metalloproteinases; proteinase-inhibitor complex; X-ray crystal structure Introduction Tumor necrosis factor-α (TNF-α), a major immu- nomodulatory 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 metallopro- teinase” (ADAM) family and the metzincin super- family, 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, *Corresponding author. E-mail address: bode@biochem.mpg.de. Abbreviations used: ADAM, a disintegrin and metalloproteinase; ADAMTS, ADAMs with thrombospondin motifs; β-APP, amyloid precursor protein; cd, catalytic domain; ECM, extracellular matrix; MMP, matrix metalloproteinase; TACE, TNF-α converting enzyme; TIMP, tissue inhibitor of metalloproteinases; N- TIMP, N-terminal domain of TIMP; TNF-α, tumor necrosis factor-α; VAP, vascular apoptosis-inducing protein. doi:10.1016/j.jmb.2008.06.088 J. Mol. Biol. (2008) 381, 1307–1319 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.