Exploring the RING-Catalyzed Ubiquitin Transfer Mechanism by MD and QM/MM Calculations

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.

Mechanistic insights revealed by a UBE2A mutation linked to intellectual disability

“…Two mechanisms are proposed for lysine deprotonation by E2s: 1) direct abstraction of a proton by a proton acceptor and 2) reduction of the pKa of the incoming lysine by the microenvironment near the E2 active site 35 . In the former case, D117 of UBE2D1 41 and H94 of UBE2G2 42 are proposed to serve as proton acceptors for an incoming lysine. But neither of the corresponding residues in UBE2A (S120 and Q93, respectively) can serve as proton acceptors and indeed, none of the residues observed to be perturbed in Q93E- UBE2A can receive a proton.…”

A conserved asparagine in a ubiquitin‐conjugating enzyme positions the substrate for nucleophilic attack

“…It is also commonly hypothesized that the side chain of a nearby asparagine acts as an oxyanion hole and stabilizes the negative charge on the zwitterionic intermediate . There is evidence to support this claim.…”

“…The rate‐limiting step in this mechanism is unknown. However, calculated reaction profiles of thioester aminolysis on model systems and on an E2 enzyme ternary complex indicate that forming the intermediate is rate limiting …”

“…Second, if the asparagine is mutated, the catalytic rate and efficiency of ubiquitination decrease significantly . Third, as shown by X‐ray crystallography and computation, it is the only plausible residue capable of acting as an oxyanion hole. However, there have been some recent studies that have reexamined this assumption using crystallography and molecular dynamics (MD) and found evidence that the asparagine hydrogen bonds to a nearby loop in the E2 enzyme, which may affect the loop's dynamics.…”

E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft

“…Therefore, Asn85 of the H is- P ro- A sn (HPN) motif, Tyr87 and Asp127 play important roles in lowering the substrate lysine pKa to allow its nucleophilic attack of the Ube2I ~ SUMO thioester bond. Several studies have demonstrated the importance of these residues for the catalytic function of different E2s, especially the HPN motif 59 60 61 62 63 64 . Asp127 plays an important role in the catalytic cleft of Ube2I 57 , a role which is likely to be generally important in E2s.…”