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

ABSTRACT: 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-9Cat) 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-9Catvariant that is active but stable to autocleavage. For this purpose, we first identified potential autocleavage sites on MMP-9Cat using mass spectroscopy and then eliminated the autocleavage site by predicting mutations that minimize autocleavage potential without reducing enzyme stability. Four computationally designed MMP-9Cat 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-9Cat variant, with an identical to MMP-9Cat WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9Cat stabilization could be applied to redesign of other proteases to improve their stability for various biotechnological applications.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Solomon Oguche  

LAB HEAD: Julia M shifman

PROVIDER: PXD041767 | Pride | 2023-10-24

REPOSITORIES: Pride