Gelatin-binding Region of Human Matrix Metalloproteinase-2

Abstract

Human matrix metalloproteinase-2 (MMP-2) contains an array of three fibronectin type II (FII) modules postulated to interact with gelatin (denatured collagen).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); characterize its ligand-binding properties; and derive dynamics properties and relative 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 possessing a putative collagen-binding site. Interaction of the synthetic gelatinlike octadecapeptide (Pro-Pro-Gly) 6 (PPG6) with COL-3 is weaker than with COL-2. We found that a synthetic peptide comprising segment 33-42 (peptide 33-42) 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 prefers peptide 33-42 over PPG6, which suggests that intramolecular interactions with the prodomain could modulate binding of pro-MMP-2 to its gelatin substrate. In COL-23, the two modules retain their structural individuality and tumble independently. Overall, the NMR data indicate that the relative orientation of the modules in COL-23 is not fixed in solution, that the modules do not interact with one another, and that COL-23 is rather flexible. The binding sites face opposite each other, and their responses to, and normalized affinities for, the longer ligand PPG12 are virtually identical to those of the individual domains for PPG6, thus precluding cooperativity, although they may interact simultaneously with multiple sites of the extracellular matrix. (2) and, unique among the metalloproteinases, three in-tandem fibronectin type II (FII) modules, which are inserted in the catalytic domain in the vicinity of the active site. In its latent form, the prodomain folds over the active-site cleft and contributes a cysteine thiol group, which coordinates the catalytic zinc ion and, as indicated by the recent x-ray crystallographic model (3), inserts the side chain of Phe 37 into the hydrophobic pocket of the third FII domain. This interaction can be disrupted by proteolysis. Once the active site is free, MMP-2 undergoes autolytic cleavage, resulting in loss of the prodomain (1).The FII modules account for the affinity of MMP-2 for gelatin, type I and IV collagens, elastin, and laminin (4 -9). A number of residues involved in binding of small hydrophobic ligands to the related second FII module of the bovine seminal fluid protein PDC-109 (PDC-109/b) were inferred from 1 H NMR studies (10). In addition, several residues that are important for interaction with gelatin have been identified, via site-directed mutagenesis, in the second FII modules from MMP-2 (11) and MMP-9 (12). However, little is known as to how tandem arrays of FII domains interact with other molecules. Fragments containing two or three consecutive FII modules from MMP-2 exhibit significantly higher apparent affinities for immobilized gelatin than any of the single modules (5...

Peptide Ligands for the Fibronectin Type II Modules of Matrix Metalloproteinase 2 (MMP-2)

“…Additionally, the ligand binding surfaces of all three modules of MMP-2 have been mapped from 1 H and 15 N NMR perturbations induced by (PPG) 6 and the longer chain analog, (PPG) 12 , synthetic peptide mimics of gelatin (2-4). In line with the crystallographic evidence, which shows that the FN2 modules in MMP-2 point away from each other (5), our NMR studies of the interaction between Col domains and (PPG) 6 and (PPG) 12 have shown that consecutive Col modules contain distinct ligand-binding sites in which affinities for these ligands are virtually identical to those of the individual domains (3,4,8).Although the affinity of the MMP-2 Col domains for collagenous ligands appears by now to be well established, less is known regarding the specificity of the interaction. In our previous studies we found that the peptide PIIKFPGDVA, which corresponds to segment 33-42 of the pro-MMP-2, interacts with the three Col domains of MMP-2 in a manner that mimics the interaction with the collagen-like (PPG) 6 and (PPG) 12 peptides (3, 4).…”

“…The latter may account for the competition between these peptides and gelatin for binding to FN2 domains. Interestingly, residues Gly-33 and Arg-34 are also involved in intramolecular interactions between the Col-3 module and the propeptide domain (3,5).…”

“…In our previous studies we found that the peptide PIIKFPGDVA, which corresponds to segment 33-42 of the pro-MMP-2, interacts with the three Col domains of MMP-2 in a manner that mimics the interaction with the collagen-like (PPG) 6 and (PPG) 12 peptides (3,4). Preference for binding to Col-3 was indicated, consistent with the x-ray crystallographic structure of the pro-MMP-2 (5).…”

“…The solution conformation of each FN2 repeat from human MMP-2 has been characterized via NMR spectroscopy (2)(3)(4). Moreover, the x-ray crystallographic structure of the intact human pro-MMP-2 has been reported (5).…”

“…Additionally, the ligand binding surfaces of all three modules of MMP-2 have been mapped from 1 H and 15 N NMR perturbations induced by (PPG) 6 and the longer chain analog, (PPG) 12 , synthetic peptide mimics of gelatin (2)(3)(4). In line with the crystallographic evidence, which shows that the FN2 modules in MMP-2 point away from each other (5), our NMR studies of the interaction between Col domains and (PPG) 6 and (PPG) 12 have shown that consecutive Col modules contain distinct ligand-binding sites in which affinities for these ligands are virtually identical to those of the individual domains (3,4,8).…”