Crystal structures of an E1\u2013E2\u2013ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

Lingmin Yuan,Melanie J Adams,Shaun K Olsen,Zongyang Lv

doi:10.1038/s41467-021-22598-y

Abstract

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

Highlights

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification
We focused on the S. cerevisiae E1, Uba[1], and the E2, Cdc[34], because of the defined biological importance of this enzyme pair as a key regulator of the cell cycle and due to the fact that they are amenable to crystallization
Structural studies of doubly loaded E1–E2–Ub(a)–Ub(t) complexes have been hampered by the labile nature of thioester bonds, the low affinity of E1–E2 complexes, and the transience of the intermediate formed during transthioesterification

Summary

Introduction

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. During E1-catalyzed activation, the C-terminus of Ub is adenylated and subsequently becomes linked via thioester bond to a catalytic cysteine residue on E110,11 This is followed by the recruitment of E2 conjugating enzymes and the transfer of Ub from the E1 catalytic cysteine to the E2 catalytic cysteine in a process called E1–E2 thioester transfer (or transthioesterification)[12,13,14]. Previous structural studies have shown that Ub E1 is a multidomain enzyme in which each domain plays a distinct functional role in its three catalytic activities of adenylation, thioester bond formation, and transthioesterification[15,16,17,18,19,20,21,22]. The Ubfold domain (UFD), which has recently been identified as a potential druggable site for cancer treatment[23] is involved in molecular recognition of E2s and subsequently in the transfer of

Methods

Results

Conclusion