The gelatin-binding site of the second type-II domain of gelatinase A MMP-2

We have shown previously that all three fibronectin type-II modules of gela tinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to loca lize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. thei r resistance to thermal and chaotropic agent-induced denaturation, was char acterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant eff ect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mut ant Y37A, the structure and stability of the mutant protein is similar to t he wild-type module. However, its gelatin affinity was severely impaired co mpared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture s uggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PD C-109 places Y37 on the right-hand rim of a hydrophobic pocket that include s residues F20, W39, Y46, Y52 and F54, and thus provides proof for the invo lvement of this pocket in ligand binding.