ALANINE SCANNING MUTAGENESIS AND FUNCTIONAL-ANALYSIS OF THE FIBRONECTIN-LIKE COLLAGEN-BINDING DOMAIN FROM HUMAN 92-KDA TYPE-IV COLLAGENASE

The human 72-kDa (CLG4A) and 92-kDa (CLG4B) type IV collagenases conta in a domain consisting of three contiguous copies of the fibronectin ( FN)-derived type II homology unit (T2HU), T2HU-1, T2HU-2, and T2HU-3. To investigate the functional role of this domain, we have constructed plasmids expressing beta-galactosidase fusion proteins with one or mo re of the CLG4B-derived T2HU. The gelatin binding assays demonstrate t hat a single copy of T2HU-2 renders beta-galactosidase capable of bind ing gelatin. The three repeats, however, differ dramatically in their capacity to bind gelatin, with T2HU-1 and T2HU-3 having significantly less binding activity than T2HU-2. Using alanine scanning mutagenesis we have defined the amino acid residues (Arg307, Asp309, Asn319, Tyr32 0, Asp323) that are critical for gelatin binding of T2HU-2. The low ge latin binding of T2HU-1 compared to T2HU-2 was traced to the non-conse rved residues Ala228-Ala and Leu253-Pro. The results suggest that the gelatin binding of the type IV collagenase proenzyme is mediated by th e FN-like domain, although the presence of another gelatin-binding sit e cannot be excluded. The FN domain-mediated binding, however, is not a rate-limiting step in the hydrolysis of gelatin by the enzyme.