Abstract
Ubiquitylation is a universal mechanism for controlling cellular functions. A large family of ubiquitin E3 ligases (E3) mediates Ubiquitin (Ub) modification. To facilitate Ub transfer, RING E3 ligases bind both the substrate and ubiquitin E2 conjugating enzyme (E2) linked to Ub via a thioester bond to form a catalytic complex. The mechanism of Ub transfer catalyzed by RING E3 remains elusive. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations, we characterized this catalytic mechanism in detail. The three-dimensional model of dimeric RING E3 ligase RNF4 RING, E2 ligase UbcH5A, Ub and the substrate SUMO2 shows close contact between the substrate and Ub transfer catalytic center. Deprotonation of the substrate lysine by D117 on UbcH5A occurs with almost no energy barrier as calculated by MD and QM/MM calculations. Then, the side chain of the activated lysine gets close to the thioester bond via a conformation change. The Ub transfer pathway begins with a nucleophilic addition that forms an oxyanion intermediate of a 4.23 kcal/mol energy barrier followed by nucleophilic elimination, resulting in a Ub modified substrate by a 5.65 kcal/mol energy barrier. These results provide insight into the mechanism of RING-catalyzed Ub transfer guiding the discovery of Ub system inhibitors.
Highlights
Protein modifications by the addition of Ub or ubiquitin-like proteins have emerged as major mechanisms that control diverse biological processes in the cell [1], and in developing tissues [2]
Subsequent quantum mechanics/molecular mechanics (QM/MM) simulations were performed to investigate the mechanism of Ub transfer to scan the potential energy for interactions between the substrate and enzyme throughout the process
The Ub transfer process is triggered through the covalent thioester bonding of Ub and E1
Summary
Protein modifications by the addition of Ub or ubiquitin-like proteins have emerged as major mechanisms that control diverse biological processes in the cell [1], and in developing tissues [2]. E1 forms a covalent thioester intermediate with the C terminus of Ub in an ATP-dependent manner. Ub is transferred from E1 to E2 via a transthiolation reaction resulting in a covalent thioester linkage between E2’ catalytic cysteine and Ub’s C terminus (E2,Ub). E3s promote the transfer of Ub from E2 to the substrate lysine amino group. There are two domains in E3s binding E2 and substrate to facilitate this process. E1 and E3 binding sites on E2s substantially overlap, so E2s dissociate from ligase domains to be ‘‘reloaded’’ with Ub [8,9,10]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
