Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly ( Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triplehelical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.Collagen catabolism is considered a committed physiological event in remodeling of tissues and embryonic development as well as a critical step in the pathology of numerous diseases (1-3). The triple-helical structure of collagen renders it resistant to most proteases. Several members of the metallopeptidase "clan" MA(M), specifically matrix metalloproteinases (MMPs), 2 possess collagenolytic activity (4). For example, one or more of the interstitial collagens (types I-III) are hydrolyzed within their triple-helical domain by MMP-1, -2, -8, -13, -14, and -18 (5). Other members of the MMP family, such as MMP-3, share similar primary and tertiary structures and substrate sequence specificities with collagenolytic MMPs but do not cleave triple-helical structures (6 -9). Triple-helical structure itself provides favorable interactions with several MMPs (i.e. MMP-1 and MMP-8 hydrolyze a triple-helical substrate more efficiently than an analogous single-stranded one), whereas the activities of other MMPs, such as MMP-3, are reduced when susceptible sequences are incorporated within a triple-helical structure (9 -12). Considerable evidence has accumulated that non-catalytic domains or regions beyond the active site, termed exosites, play significant roles in MMP recognition and processing of natural protein substrates (8,(13)(14)(15). The apparent regulatory role played by the collagen triple helix, in concert with recognition by regions beyond the protease active site, suggests that topology of the substrate may be an element of proteolytic specificity.In the present study we considered the topological specificity of the metallopeptidase family M12(B), which are members of the same MA(M) clan as MMPs but are not known to cleave triple-helical domains within collagens. More specifically, we examined the a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family to evaluate the role of substrate topology on enzyme activity. Among the ADAMTS family members, several have been described to have proteolytic activity toward aggrecan (ADAM...