Matrix metalloproteinase-9 (MMP-9) is a protease that remodels the extracellular matrix and has been implicated in many diseases including neurodegeneration, arthritis, cardiovascular and fibrosis and a major driver of cancer metastasis. Hence, there is a high demand for MMP-9 inhibitors for therapeutic purposes and many research labs and companies are working on designing drugs that target this enzyme. For such drug design efforts, large amounts of MMP-9 are required. Yet, MMP-9 is an intrinsically unstable protein that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. To alleviate self-cleavage and improve MMP-9 stability, MMP-9 mutants that remove the catalytic Glu and abolish catalytic activity have been utilized in some studies. Such enzyme variants are more stable but present a modified active site, and thus could not be used in MMP-9 drug design or screening experiments. 

Alessandro Bonadio and Solomon Oguche, a Ph. D. and an M. Sc. students from the Shifman lab, set their goal to design MMP-9 variant that is stable to autocleavage and yet does not loose enzyme activity. For this purpose, the students developed a novel approach that first identifies the auto-cleavage site using the mass spectrometry and then removes this site from the protein sequences. Using computational design, they introduced a minimum number of mutations that preserve protein structure and activity but decrease the probability of sequence cleavage by MMP-9. The best designed MMP-9 variant with two mutations 10 Å away from the active site does not auto-cleave after seven days of incubation at 37°C while retaining WT-like MMP-9 activity. Our work has proved that removal of auto-cleavage sites could result in production of proteases with incredible stability. Such enzyme is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9 stabilization could be applied to redesign of other proteases to improve their stability for various biotechnological applications.

Read the paper https://portlandpress.com/biochemj/article/480/14/1097/233252/Computational-design-of-Matrix-Metalloproteinase-9