Computational design of Matrix Metalloprotenaise-9 (MMP-9) resistant to autocleavage

Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix and has been implicated as a major driver in cancer metastasis. Hence, there is a high demand for MMP-9 inhibitors for therapeutic purposes. For such drug design efforts, large amounts of MMP-9 are required. Yet, the catalytic domain of MMP-9 (MMP-9(Cat)) is an intrinsically unstable enzyme that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. We set our goal to design MMP-9(Cat) variant that is active but stable to autocleavage. For this purpose, we first identified potential autocleavage sites on MMP-9(Cat) using mass spectroscopy and then eliminated the autocleavage site by predicting mutations that minimize autocleavage potential without reducing enzyme stability. Four computationally designed MMP-9(Cat) variants were experimentally constructed and evaluated for auto-cleavage and enzyme activity. Our best variant, Des2, with 2 mutations, was as active as the wild-type enzyme but did not exhibit auto-cleavage after seven days of incubation at 37°C. This MMP-9(Cat) variant, with an identical to MMP-9(Cat) WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9(CAT) stabilization could be applied to redesign of other proteases to improve their stability for various biotechnological applications.