Structural Basis for Autoinhibition and Mutational Activation of Eukaryotic Initiation Factor 2α Protein Kinase GCN2*[boxs]

Alan G. Hinnebusch,Stephen K. Burley,Anil K. Padyana,Hongfang Qiu,Antonina Roll-Mecak

doi:10.1074/jbc.m504096200

Abstract

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Highlights

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2
The isolated protein kinase (PK) domain of GCN2 is completely inert in vitro, but, remarkably, activity is rescued by single amino acid substitutions at Arg794 (R794G) or Phe842 (F842L). These constitutively activating (GCN2c) mutations bypass the tRNA binding requirement for kinase activation in vivo, and we proposed previously that they alter the PK active site in a way that mimics conformational changes induced by interactions with the histidyl-tRNA synthetase (HisRS) or ribosomebinding and dimerization domain (RB/DD) domains on tRNA binding [24]
We propose a two-step activation mechanism in which tRNA binding to the HisRS domain leads to a comparable structural remodeling of the hinge region of wild-type GCN2 that facilitates ATP binding

Summary

Introduction

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. Mammalian and yeast cells respond to starvation or stress by down-regulating overall protein synthesis, while increasing translation of specific mRNAs encoding transcription factors responsible for ameliorating starvation or stress conditions. These responses are induced by protein kinases that phosphorylate serine 51 in the ␣-subunit of translation initiation factor 2 (eIF2␣).. A ribosomebinding and dimerization domain (RB/DD) at the extreme C tion factor 2; PK, protein kinase; HisRS, histidyl-tRNA synthetase; PDB, Protein data bank; r.m.s.d., root mean square deviation; cAPK, cAMP dependent protein kinase; CDK2, cyclin-dependent kinase 2; c-Src, tyrosine kinase c-Src; CAPSO, N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid; CHES, N-cyclohexyl-2-aminoethanesulfonic acid; RB/DD, ribosome-binding and dimerization domain; AMPPNP, 5Ј-adenylyl-␤,␥-imidodiphosphate; WT, wild-type; GCN2, general control non-derepressible-2

Results

Discussion

Conclusion