Structure and domain-domain interactions of the gelatin binding site of human 72-kilodalton type IV collagenase (gelatinase A, matrix metalloproteinase 2).

We have shown previously that all three fibronectin type II modules of gelatinase A contribute to its gelatin affinity. In the present investigation we have studied the structure and module-module interactions of this gelatin-binding domain by circular dichroism spectroscopy and differential scanning calorimetry. Comparison of the Tm values of the thermal transitions of isolated type II modules with those of bimodular or trimodular proteins has shown that the second type II module is significantly more stable in the trimodular protein coll 123 (Tm = 54 degrees C) than in the single-module protein coll 2 (Tm = 44 degrees C) or in the bimodular proteins coll 23 (Tm = 47 degrees C) and coll 12 (Tm = 48 degrees C). Analysis of the enthalpy changes associated with thermal unfolding of the second type II module suggests that it is stabilized by domain-domain interactions in coll 123. We propose that intimate contacts exist between the three tandem type 11 units and they form a single gelatin-binding site. Based on the three-dimensional structures of homologous metalloproteases and type II modules, a model is proposed in which the three type II units form an extension of the substrate binding cleft of gelatinase A.