Analysis of interactions in the regulatory complex eIF2:eIF2B governing protein synthesis

Abstract

Any living cell relies on translation to fuel its metabolism and perform essential functions. Eukaryotic initiation factor 2 (eIF2) is a heterotrimeric GTPase that lies at the core of protein synthesis, being responsible to load the ribosome with methionyl tRNA initiator (Met-tRNAi) at each round of translation. The functional cycle of eIF2 depends on additional factors including eIF2B and eIF5. These proteins have antagonist roles and compete for mutually exclusive interfaces of eIF2. As part of the integrated stress response, eIF2 is phosphorylated and traps all available eIF2B in an inhibitory interaction that results in the global shutdown of protein synthesis. Here, we gather insights into the molecular mechanism of eIF2 and eIF2B, and their interaction with eIF5 and the Met-tRNAi. We present the cryo-EM structures of phosphorylated and unphosphorylated yeast eIF2 in complex with eIF2B, revealing one of their two binding modes. Through structural modelling and computational analysis, we identify conserved elements within eIF2 that rearrange upon binding to two different pockets of eIF2B. Their role is addressed by complementary genetic and biochemical approaches. Consequently, we propose that phosphorylation constrains eIF2 to adopt a conformation that stably binds the regulatory site of eIF2B, preventing the dissociation of the complex. Finally, we advance a structural model of eIF2 recycling consisting of consecutive coupled interactions of eIF2 with eIF5, eIF2B and Met-tRNAi.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Graham Pavitt (Supervisor), Martin Pool (Supervisor) & Alan Roseman (Supervisor)