TY - JOUR
T1 - Alanine scanning mutagenesis and functional analysis of the fibronectin- like collagen-binding domain from human 92-kDa type IV collagenase
AU - Collier, I. E.
AU - Krasnov, P. A.
AU - Strongin, A. Y.
AU - Birkedal-Hansen, H.
AU - Goldberg, G. I.
PY - 1992
Y1 - 1992
N2 - 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 β- 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 β- 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.
AB - 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 β- 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 β- 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.
UR - http://www.scopus.com/inward/record.url?scp=0026656315&partnerID=8YFLogxK
M3 - Article
C2 - 1313021
AN - SCOPUS:0026656315
VL - 267
SP - 6776
EP - 6781
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 10
ER -