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. 2014 Jul 8;9(7):e101663.
doi: 10.1371/journal.pone.0101663. eCollection 2014.

Exploring the RING-catalyzed ubiquitin transfer mechanism by MD and QM/MM calculations

Yunmei Zhen  1 , Guangrong Qin  2 , Cheng Luo  2 , Hualiang Jiang  2 , Kunqian Yu  2 , Guanghui Chen  3
Affiliations

Exploring the RING-catalyzed ubiquitin transfer mechanism by MD and QM/MM calculations

Yunmei Zhen et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Interactions between UbcH5A (E2) and SUMO2 (sub) in the R2 trajectory.
E2 UbcH5A is shown in cyan, E3 RNF4 is shown in green, Ub is shown in magenta, and substrate SUMO2 is shown in yellow. (A–C) Detail of interactions between E2 UbcH5A (cyan) and substrate SUMO2 (yellow). (D) Hydrogen bonds occupancies during production MD. (E) Hydrophobic interactions occupancies during production MD.
Figure 2
Figure 2. Overview of the nucleophilic attack.
(A) The potential energy surface of nucleophilic attack along defined reaction coordinates. (B) The relative energy of representative conformations along the reaction pathway. (C) The QM/MM optimized structures of the reactant (R), transition states (TSs), transition immediate (TIs), and product (P).
Figure 3
Figure 3. Interactions between enzyme (E2 and E3) and substrate (sub) in the R2_H trajectory and comparison of the R2 system.
E2 UbcH5A is shown in cyan, E3 RNF4 is shown in green, Ub is shown in magenta, and substrate SUMO2 is shown in yellow. (A) Structure detail of the interaction between E2 (UbcH5A) and substrate (SUMO2) in the E2 active site. (B) Detail of the substrate (SUMO2) and ligase interaction. (C) Hydrogen bond occupancies in R2_H compared to R2. (D) Hydrophobic interaction occupancies of both R2 and R2_H systems.
Figure 4
Figure 4. Two possible deprotonation pathways of lysine through D117.
(A) Average structure (A′) in the first cluster in R2_H MD simulations and its optimized conformation (A″). (B) The second clustered structure (B′) and its optimized conformation (B″).
Figure 5
Figure 5. The proposed RING-catalyzed Ub transfer mechanism.
See this image and copyright information in PMC

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Grants and funding

This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (No. 2012AA020301 and 2012AA01A305), the National Natural Science Foundation of China grants (81230076 and 21210003), Chinese Academy of Sciences (Project KSZD-EW-L09-4). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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