Catalytic Mechanism of the Ubiquitin-Like NEDD8 Transfer in RING E3-E2∼NEDD8-Target Complex from QM/MM Free Energy Simulations

Ubiquitin-like (UBL) protein modifications play a key role in regulating protein function. In contrast to the ubiquitin (UB) and small ubiquitin-like modifier (SUMO) which are ligated to a massive segment of proteome, the UBL NEDD8 is highly selective for modifying a lysine residue on closely related cullin proteins (CULs). In this study, the X-ray structure of a trapped E3-E2 similar to NEDD8-target intermediate (RBX1-UBC1 similar to NEDD8-CUL1-DCN1) is used to build computer models, and combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) and free energy (potential of mean force) simulations are performed to investigate the catalytic mechanism of the NEDD8 transfer from E2 to the lysine residue (K720) on the substrate in the complex. The role of the active site residues is examined. The simulation results show that either E117 or D143 from E2 may be able to work as a general base catalyst to deprotonate K720 on the substrate, and K720 can then perform the nucleophilic attack on the thioester bond linking E2 and NEDD8. It is also shown that the formation of a new isopeptide bond between K720 and NEDD8 and the breaking of the thioester bond are concerted based on the computer simulations. Furthermore, the results suggest that K720 may act as a general acid catalyst to protonate the leaving group of C111 from E2. The free energy barrier for nucleophilic attack is estimated to be 14-15 kcal/mol based on the free energy simulations.