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 contain 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 more of the CLG4B-derived T2HU. The gelatin binding assays demonstrate that a single copy of T2HU-2 renders beta-galactosidase capable of binding 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, Tyr320, Asp323) that are critical for gelatin binding of T2HU-2. The low gelatin binding of T2HU-1 compared to T2HU-2 was traced to the non-conserved residues Ala228-Ala and Leu253-Pro. The results suggest that the gelatin binding of the type IV collagenase proenzyme is mediated by the FN-like domain, although the presence of another gelatin-binding site cannot be excluded. The FN domain-mediated binding, however, is not a rate-limiting step in the hydrolysis of gelatin by the enzyme.
Collier
I E
IE
Division of Dermatology, Washington University School of Medicine, St. Louis, Missouri 63110.
Krasnov
P A
PA
Strongin
A Y
AY
Birkedal-Hansen
H
H
Goldberg
G I
GI
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism; D006801:Humans; D003012:Microbial Collagenase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D001616:beta-Galactosidase / Q000378:metabolism
D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D005780:Gelatin; D001616:beta-Galactosidase; D003012:Microbial Collagenase; D000409:Alanine; D004121:Dimethyl Sulfoxide