Substrate Conformation Modulates Aggrecanase (ADAMTS-4) Affinity and Sequence Specificity

• Published in: The Journal of biological chemistry 2007-01, Vol.282 (1), p.142-150
• Main Authors: Lauer-Fields, Janelle L., Minond, Dmitriy, Sritharan, Thilaka, Kashiwagi, Masahide, Nagase, Hideaki, Fields, Gregg B.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M605236200

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(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with Km values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient “triple-helical peptidase” activity and fell into one of two categories: (kcat/Km)SSP > (kcat/Km)THP (thermolysin, trypsin, and MMP-13) or (kcat/Km)THP ≥ (kcat/Km)SSP and (Km)SSP > (Km)THP (MMP-1 and ADAMTS-4). 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.