Functional basis for the overlap in ligand interactions and substrate specificities of matrix metalloproteinases-9 and -2

The MMPs (matrix metalloproteinases) MMP-9 and -2 each possess a unique CBD (collagen-binding domain) containing three fibronectin type II-like modules. The present experiments investigated whether the contributions to ligand interactions and enzymatic activities by the CBD of MMP-9 (CBD-9) corresponded to those of CBD in MMP-2 (CBD-2). The interactions of recombinant CBD-9 with a series of collagen types and extracellular matrix molecules were characterized by protein-protein binding assays. CBD-9 bound native and denatured type I, II, III, IV and V collagen, as well as Matrigel and laminin, with apparent Kd values of (0.1-6.8) × 10^-7 M, which were similar to the K_d values for CBD-2 [(0.2-3.7) × 10^-7 M]. However, CBD-9 bound neither native nor denatured type VI collagen. We also generated two modified MMPs, MMP-9^E402A and MMP-2^E404A, by site-specific mutations in the active sites to obtain enzymes with intact ligand binding, but abrogated catalytic properties. In subsequent competitive binding assays, CBD-9 and MMP-9^E402A inhibited the interactions of MMP-2^E404A and, conversely, CBD-2 and MMP-2^E404A competed with MMP-9 ^E402A binding to native and denatured type I collagens, pointing to shared binding sites. Importantly, the capacity of CBD-9 to disrupt the MMP-9 and MMP-2 binding of collagen translated to inhibition of the gelatinolytic activity of the enzymes. Collectively, these results emphasize the essential contribution of CBD-9 to MMP-9 substrate binding and gelatinolysis, and demonstrate that the CBDs of MMP-9 and MMP-2 bind the same or closely positioned sites on type I collagen.