Identification of the (183)RWTNNFREY(191) region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity

• Published in: The Journal of biological chemistry 2000-09, Vol.275 (38), p.29610-29617
• Main Authors: Chung, L, Shimokawa, K, Dinakarpandian, D, Grams, F, Fields, G B, Nagase, H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites - genetics; CHO Cells; Collagen - metabolism; Cricetinae; Enzyme Activation - genetics; Humans; Matrix Metalloproteinase 1 - genetics; Matrix Metalloproteinase 1 - metabolism; Molecular Sequence Data; Protein Conformation; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Structure-Activity Relationship; Substrate Specificity - genetics
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M004039200

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.