Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity. Suggestion of a common topological specificity for functionally diverse proteases.
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 K(m) 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: (k(cat)/K(m))(SSP) > (k(cat)/K(m))(THP) (thermolysin, trypsin, and MMP-13) or (k(cat)/K(m))(THP) > or = (k(cat)/K(m))(SSP) and (K(m))(SSP) > (K(m))(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 triple-helical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.
Lauer-Fields
Janelle L
JL
Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, Florida 33431-0991, USA.
Minond
Dmitriy
D
Sritharan
Thilaka
T
Kashiwagi
Masahide
M
Nagase
Hideaki
H
Fields
Gregg B
GB
D051722:ADAM Proteins / Q000737:chemistry* / Q000378:metabolism; D000071121:ADAMTS4 Protein; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D020781:Matrix Metalloproteinase 1 / Q000737:chemistry; D053509:Matrix Metalloproteinase 13 / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011348:Procollagen N-Endopeptidase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
D010455:Peptides; D051722:ADAM Proteins; D053509:Matrix Metalloproteinase 13; D011348:Procollagen N-Endopeptidase; D020781:Matrix Metalloproteinase 1; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human