The recognition of collagen and triple-helical Toolkit peptides by MMP-13: Sequence specificity for binding and cleavage [Protein Synthesis and Degradation]

July 9th, 2014 by Howes, J.-M., Bihan, D., Slatter, D. A., Hamaia, S. W., Packman, L. C., Knauper, V., Visse, R., Farndale, R. W.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly775-Leu776 in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site (at helix residues 775-6). MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N-terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.
  • Posted in Journal of Biological Chemistry, Publications
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