Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13.
The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.
O'Shea
M
M
Strangeways Research Laboratory, Cambridge, U.K.
Willenbrock
F
F
Williamson
R A
RA
Cockett
M I
MI
Freedman
R B
RB
Reynolds
J J
JJ
Docherty
A J
AJ
Murphy
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D019714:Tissue Inhibitor of Metalloproteinases; D014162:Transfection; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D003545:Cysteine