Validation of catalytic site residues of Ubiquitin Specific Protease 2 (USP2) by molecular dynamic simulation and novel kinetics assay for rational drug design.

Post-translational modifications of proteins such as protein ubiquitination are crucial for regulating conformation, stability and localization of the modified protein. Ubiquitin-specific protease 2 (USP2), a multifunctional cysteine protease is reported to be a key regulator of ubiquitylation events in numerous oncogenic proteins e.g., fatty acid synthetase, Mdm2, EGFR, cyclin A1, and cyclin-D1, etc. Thus targeting USP2 is a promising strategy for cancer therapy. USP2 is characterized by a catalytic triad comprising of cysteine, histidine and aspartic acid residues. Five residues including three from the catalytic triad and two from outside of the catalytic triad have been reported as a catalytic site of USP2 that catalyze hydrolysis and stabilizes the oxyanion formed in the intermediate step of catalysis. Here, we report two more novel residues (L269 and Y558) on USP2 involved in the catalysis of Ubiquitin using computational alanine scanning (CAS) followed by molecular dynamic simulation studies. The results obtained from CAS were further validated by a highly reliable, time- and cost-effective SDS-PAGE-based kinetics assay using UBA52 which is a natural substrate of USP2. Our results showed that mutating L269 and Y558 significantly compromised the catalytic efficiency of USP2 in hydrolyzing UBA52 which can further be extended to rational drug design of USP2 selective inhibitors and to explore the catalytic sites of other USPs. Two novel residues take part in catalytic activity of USP2 which were depicted by MD Simulations and were further validated by novel SDS-PAGE-based reliable time- and cost-effective kinetics assay.