Identification of the (RWTNNFREY191)-R-183 region as a critical segment ofmatrix metalloproteinase 1 for the expression of collagenolytic activity

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen tr iple helices into 3/4 and 1/4 fragments. To understand the structural eleme nts responsible for this activity, various lengths of MMP-1 segments have b een 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, r ecombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residue s (RWTNNFREY191)-R-183 located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expressi on of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the co rresponding 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 substr ates, 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 n ot express any collagenolytic activity. It is therefore concluded that RWTN NFREY, together with the C-terminal hemopexin domain, is essential for coll agenolytic activity but that additional structural elements in the catalyti c domain are also required. These elements probably act in a concerted mann er to cleave the collagen triple helix.