Abstract
Matrix metalloproteinase 2 (MMP-2) contains three fibronectin type II (col) modules that contribute to its collagen specificity. We observed that the CD spectra of the separate col modules account for the CD and temperature profiles of the in-tandem col-123 construct. Thus, to the extent of not significantly perturbing the secondary structure and thermal stability characteristics of the neighboring units, the domains within col-123 do not interact. Via NMR, we investigated ligand binding properties of the three repeats within col-123: col-123/1 (the col-1 domain within col-123), col-123/2, and col-123/3. Interactions of col-123 with the collagen mimic peptide (Pro-Pro-Gly)6 (PPG6) and propeptide segment PIIKFPGDVA (p33-42) were studied. While col-123/1 and col-123/2 bound PPG6, they interacted more weakly with p33-42. In contrast, col-123/3 exhibited a higher affinity for p33-42 than for PPG6. Thus, despite their structural homology, the col repeats of MMP-2 differ in substrate specificity. Furthermore the binding affinities toward the three in-tandem col repeats were close to those determined for the individual isolated domains or for col-12/1, indicating that vis-a-vis these ligands each module interacts essentially as an autonomous unit. Interestingly the domains within col-123 exhibited enhanced affinities for Hel3, a construct that contains ((Gly-Pro-Pro)12)3 in triple helical configuration. Nevertheless the affinities were significantly higher for col-123/1 and col-123/2 relative to col-123/3 in line with their behaviors toward PPG6. This hints at a cooperative participation toward Hel3, which is a closer mimic of collagen, a hypothesis that is supported by the detected lower affinities of col-12/1, col-12/2, col-2, col-23/2, col-3, and col-23/3 for Hel3.
Highlights
Matrix metalloproteinase 2 (MMP-2) contains three fibronectin type II modules that contribute to its collagen specificity
matrix metalloproteinase (MMP)-2; col-23, the in-tandem second and third FN2 domains from human MMP-2; col-23/2, the col-2 repeat in col-23; col-23/3, the col-3 repeat in col-23; col-3, the third FN2 domain from human MMP-2; CTT, cyclic peptide CTTHWGFTLC; ⌬HvH, van’t Hoff enthalpy; ⌬SvH, van’t Hoff entropy; FN2, fibronectin type II; GPP12, the collagen mimic peptide (Gly-Pro-Pro)12; PPG12, the collagen mimic peptide (Pro-ProGly)12; Hel3, capped molecule containing peptide (GPP12)3 in triple helical configuration; PPG6, the collagen mimic peptide (Pro-Pro-Gly)6; Ka, equilibrium association constant; pro-MMP-2, the proenzyme form of MMP-2; p33– 42, peptide PIIKFPGDVA corresponding to segment
Expression and Purification of col Domains—15N-Labeled col-1, col-2, col-3, col-12, and col-23 belonged to batches described previously (16 –18). 15N-Labeled col-123 was expressed in Escherichia coli according to the protocol used for col-2 (16)
Summary
Matrix metalloproteinase 2 (MMP-2) contains three fibronectin type II (col) modules that contribute to its collagen specificity. The binding affinities toward the three in-tandem col repeats were close to those determined for the individual isolated domains or for col12/1, indicating that vis-a -vis these ligands each module interacts essentially as an autonomous unit. We report on a study of the ligand binding properties of MMP-2; col-23, the in-tandem second and third FN2 domains from human MMP-2; col-23/2, the col-2 repeat in col-23; col-23/3, the col-3 repeat in col-23; col-3, the third FN2 domain from human MMP-2; CTT, cyclic peptide CTTHWGFTLC; ⌬HvH, van’t Hoff enthalpy; ⌬SvH, van’t Hoff entropy; FN2, fibronectin type II; GPP12, the collagen mimic peptide (Gly-Pro-Pro); PPG12, the collagen mimic peptide (Pro-ProGly); Hel, capped molecule containing peptide (GPP12) in triple helical configuration; PPG6, the collagen mimic peptide (Pro-Pro-Gly); Ka, equilibrium association constant; pro-MMP-2, the proenzyme form of MMP-2; p33– 42, peptide PIIKFPGDVA corresponding to segment
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