title: The recognition of collagen and triple-helical toolkit peptides by MMP-13: sequence specificity for binding and cleavage.
abstract: 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 Gly(775)-Leu(776) 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. 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.
authors.affiliation: From the Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
authors.forename: Joanna-Marie
authors.initials: JM
authors.lastname: Howes
authors.affiliation: From the Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
authors.forename: Dominique
authors.initials: D
authors.lastname: Bihan
authors.affiliation: From the Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Slatter
authors.affiliation: From the Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
authors.forename: Samir W
authors.initials: SW
authors.lastname: Hamaia
authors.affiliation: From the Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
authors.forename: Len C
authors.initials: LC
authors.lastname: Packman
authors.affiliation: the Cardiff University Dental School, Dental Drive, Cardiff CF14 4XY, United Kingdom, and.
authors.forename: Vera
authors.initials: V
authors.lastname: Knauper
authors.affiliation: the Kennedy Institute of Rheumatology, Hammersmith, London W6 8LH, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Visse
authors.affiliation: From the Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom, rwf10@cam.ac.uk.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D024043:Collagen Type II / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D053509:Matrix Metalloproteinase 13 / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D024043:Collagen Type II; D017931:DNA Primers; D010455:Peptides; D053509:Matrix Metalloproteinase 13
keywords: Collagen; Collagenolysis; Hemopexin Domain; MMP-13; Matrix Metalloproteinase (MMP); Protease; Protein Conformation; Substrate Specificity; Toolkit Peptides