Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2)

摘要: Abstract An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these catalytically required substrate binding sites (exosites) that are located outside catalytic cleft. In MMP-2, positioning collagen molecules occurs via a unique fibronectin-like domain (CBD) contains three distinct modular sites. To characterize contributions exosites gelatinolysis by seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in fibronectin type II modules CBD. Circular dichroism spectroscopy verified recombinant wild-type (WT) had same fold. Moreover, WT activity on short FRET peptide is hydrolyzed independently CBD binding. Among single-point variants, substitution module 3 site greatest impact affinity for gelatin. Simultaneous two further reduced gelatin The rates 20–40% following substitutions, 60–75% after double-point modifications, > 90% triple-point variants. Intriguingly, contributed differentially cleavage dissociated α-1(I) α-2(I) chains. Importantly, kinetic analyses ( k cat / K m ) revealed catalysis triple-helical relied primarily site. Thus, we have identified residues essential constitute promising targets selective inhibition MMP-2.