DIFFERENT DOMAIN INTERACTIONS ARE INVOLVED IN THE BINDING OF TISSUE INHIBITORS OF METALLOPROTEINASES TO STROMELYSIN-1 AND GELATINASE-A

The matrix metalloproteinases gelatinase A and stromelysin-l have defi nable N-terminal (catalytic) and C-terminal domains. In order to analy ze their interactions with the N- and C-terminal domains of the tissue inhibitors of metalloproteinases TIMP-1 and -2, mutants of both the e nzymes and the inhibitors were prepared in which the C-terminal domain s had been deleted. Since the Ki values for TIMP inhibition of the mat rix metalloproteinases are in the picomolar range, it was not possible to measure these accurately within the sensitivity of available activ ity assays. Rate constants for the association of the wild-type protei ns were therefore determined and systematically compared with those fo r the deletion mutants. It was found that TIMP-1 binds more rapidly th an TIMP-2 to stromelysin-1 and that the C-terminal domain of the enzym e does not affect the rate of association of enzyme and inhibitor. Thi s is in contrast to gelatinase A, where the C-terminal domain has been shown to play an important role in increasing the rate of complex for mation with the TIMPs (Willenbrock et al., 1993). The TIMPs are also c omprised of an N- and C-terminal domain. By deletion mutagenesis, we f ound that the C-terminal domain of both TIMPs contributed less to the rate of complex formation with stromelysin-1 than to that with gelatin ase A. Hybrids of the N- and C-terminal domains of gelatinase A and st romelysin-1 were prepared and used to analyze further the differences in domain interactions with the TIMPs. They demonstrated that the inte ractions between the C-terminal domains of enzyme and inhibitor can oc cur irrespective of the nature of the N-terminal domain. We can conclu de that the TIMPs have two major binding regions which associate in di fferent ways with the domains of the enzymes gelatinase A and stromely sin-1. The N-terminal domains of the TIMPs bind to the enzyme catalyti c domains to inhibit activity. The TIMP C-terminal domain acts to incr ease the association rate constant by binding to the N-terminal domain of stromelysin or the C-terminal domain of gelatinase A.