Activation and Reactivity of the Deubiquitinylase\nOTU Cezanne‑2 from MD Simulations and QM/MM Calculations

Cezanne-2 (Cez2)\nis a deubiquitinylating (DUB) enzyme involved\nin the regulation of ubiquitin-driven cellular signaling and selectively\ntargets Lys11-linked polyubiquitin chains. As a representative member\nof the ovarian tumor (OTU) subfamily DUBs, it performs cysteine proteolytic\nisopeptide bond cleavage; however, its exact catalytic mechanism is\nnot yet resolved. In this work, we used different computational approaches\nto get molecular insights into the Cezanne-2 catalytic mechanism.\nExtensive molecular dynamics (MD) simulations were performed for 12\nμs to model free Cez2 and the diubiquitin (diUb) substrate-bound\nprotein–protein complex in two different charge states of Cez2,\neach corresponding to a distinct reactive state in its catalytic cycle.\nThe simulations were analyzed in terms of the relevant structural\nparameters for productive enzymatic catalysis. Reactive diUb–Cez2\ncomplex configurations were identified, which lead to isopeptide bond\ncleavage and stabilization of the tetrahedral oxyanion intermediate.\nThe reliability of these complexes was further assessed by quantum\nmechanics/molecular mechanics (QM/MM) optimizations. The results show\nthat Cez2 follows a modified cysteine protease mechanism involving\na catalytic Cys210/His367 dyad, with the oxyanion hole to be a part\nof the “C-loop,” and polarization of His367 by the formation\nof a strictly conserved water bridge with Glu173. The third residue\nhas a dual role in catalysis as it mediates substrate binding and\npolarization of the catalytic dyad. A similar mechanism was identified\nfor Cezanne-1, the paralogue of Cez2. In general, our simulations\nprovide valuable molecular information that may help in the rational\ndesign of selective inhibitors of Cez2 and closely related enzymes.