Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-alpha-converting enzyme.
We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (K(app)(i)) and association rates (k(on)) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60 pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-alpha demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-alpha. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-alpha shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.
Lee
Meng-Huee
MH
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. meng.lee@uea.ac.uk
Verma
Vandana
V
Maskos
Klaus
K
Nath
Deepa
D
Knäuper
Vera
V
Dodds
Philippa
P
Amour
Augustin
A
Murphy
Gillian
G
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D013997:Time Factors; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000737:chemistry*
D019717:Tissue Inhibitor of Metalloproteinase-3; D019859:Proto-Oncogene Proteins c-met; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D005978:Glutathione; D019791:Guanidine