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

Abstract

Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix. MMP-9 has been implicated in several diseases including neurodegeneration, arthritis, cardiovascular diseases, fibrosis and several types of cancer, resulting in 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-9Cat variant 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 other proteases to improve their stability for various biotechnological applications.

Elucidating key determinants of engineered scFv antibody in MMP-9 binding using high throughput screening and machine learning

“…Recombinant active human MMP-9cd (auto-cleavage resistance) 34 with an N-terminal His6x tag was expressed in Rosetta™(DE3) pLysS (Millipore Sigma) cells using a pET-28a(+)-His6x-MMP-9cd vector as previously described 33,35,36 . Briefly, MMP-9cd protein expression was induced with 0.5 mM IPTG for 21 hours at 25°C.…”