Activation and Reactivity of the Deubiquitinylase OTU Cezanne-2 from MD Simulations and QM/MM Calculations

Cezanne-2 (Cez2) is a deubiquitinylating (DUB) enzyme involved in the regulation of ubiquitin-driven cellular signaling and selectively targets Lys11-linked polyubiquitin chains. As a representative member of the ovarian tumor (OTU) subfamily DUBs, it performs cysteine proteolytic isopeptide bond cleavage; however, its exact catalytic mechanism is not yet resolved. In this work, we used different computational approaches to get molecular insights into the Cezanne-2 catalytic mechanism. Extensive molecular dynamics (MD) simulations were performed for 12 μs to model free Cez2 and the diubiquitin (diUb) substrate-bound protein–protein complex in two different charge states of Cez2, each corresponding to a distinct reactive state in its catalytic cycle. The simulations were analyzed in terms of the relevant structural parameters for productive enzymatic catalysis. Reactive diUb–Cez2 complex configurations were identified, which lead to isopeptide bond cleavage and stabilization of the tetrahedral oxyanion intermediate. The reliability of these complexes was further assessed by quantum mechanics/molecular mechanics (QM/MM) optimizations. The results show that Cez2 follows a modified cysteine protease mechanism involving a catalytic Cys210/His367 dyad, with the oxyanion hole to be a part of the "C-loop," and polarization of His367 by the formation of a strictly conserved water bridge with Glu173. The third residue has a dual role in catalysis as it mediates substrate binding and polarization of the catalytic dyad. A similar mechanism was identified for Cezanne-1, the paralogue of Cez2. In general, our simulations provide valuable molecular information that may help in the rational design of selective inhibitors of Cez2 and closely related enzymes.