Gelatin-binding region of human matrix metalloproteinase-2 - Solution structure, dynamics, and function of the COL-23 two-domain construct

Human matrix metalloproteinase-2 (MMP-2) contains an array of three fibrone ctin type II (FII) modules postulated to interact with gelatin (denatured c ollagen). Here, we verify that the NMR solution structure of the third FII repeat (COL-3) is similar to that of the second FII repeat (COL-2); charact erize its ligand-binding properties; and derive dynamics properties and rel ative orientation in solution for the two domains of the COL-23 fragment, a construct comprising COL-2 and COL-3 in tandem, with each domain possessin g a putative collagen-binding site. Interaction of the synthetic gelatin-li ke octadecapeptide (Pro-Pro-Gly)(6) (PPG6) with COL-3 is weaker than with C OL-2. We found that a synthetic peptide comprising segment 33-42 (peptide 3 3-42) from the MMP-2 prodomain interacts with COL-3 and, albeit with lower affinity, with COL-2 in a way that mimics PPG6 binding. COL-3 strongly pref ers peptide 33-42 over PPG6, which suggests that intramolecular interaction s with the prodomain could modulate binding of pro-MMP-2 to its gelatin sub strate. In COL-23, the two modules retain their structural individuality an d tumble independently. Overall, the NMR data indicate that the relative or ientation of the modules in COL-23 is not fixed in solution, that the modul es do not interact with one another, and that COL-23 is rather flexible. Th e binding sites face opposite each other, and their responses to, and norma lized affinities for, the longer ligand PPG12 are virtually identical to th ose of the individual domains for PPG6, thus precluding cooperativity, alth ough they may interact simultaneously with multiple sites of the extracellu lar matrix.