Structure of the catalytic fragment of translation initiation factor 2B and identification of a critically important catalytic residue.

Thomas Boesen,Sarah S Mohammad,Graham D Pavitt,Gregers R Andersen

doi:10.1074/jbc.m311055200

Thomas Boesen, Sarah S Mohammad + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m311055200

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Abstract

Eukaryotic initiation factor (eIF) 2B catalyzes the nucleotide activation of eIF2 to its active GTP-bound state. The exchange activity has been mapped to the C terminus of the eIF2Bepsilon subunit. We have determined the crystal structure of residues 544-704 from yeast eIF2Bepsilon at 2.3-A resolution, and this fragment is an all-helical protein built around the conserved aromatic acidic (AA) boxes also found in eIF4G and eIF5. The eight helices are organized in a manner similar to HEAT repeats. The molecule is highly asymmetric with respect to surface charge and conservation. One area in the N terminus is proposed to be directly involved in catalysis. In agreement with this hypothesis, mutation of glutamate 569 is shown to be lethal. An acidic belt and a second area in the C terminus containing residues from the AA boxes are important for binding to eIF2. Two mutations causing the fatal human genetic disease leukoencephalopathy with vanishing white matter are buried and appear to disrupt the structural integrity of the catalytic domain rather than interfering directly with catalysis or binding of eIF2.

Highlights

The initiation phase of protein synthesis in eukaryotic cells is a complex series of highly regulated interactions between ribosomal subunits, mRNA, aminoacylated initiator methionyltRNA (Met-tRNAiMet), and eukaryotic translation initiation factors
As we had previously identified two residues within this region of yeast eIF2B⑀ containing residues Asp544–Asp704 (eIF2B⑀C), Thr552 and Ser576, that when mutated significantly impaired eIF2B function in yeast [14] we suspected that other changes here might significantly reduce eIF2B activity
The Bipolar Properties of eIF2B⑀C—The catalytic activity of eIF2B has been mapped to residues 518 –712 by genetic analysis, in vitro exchange, and pull-down assays [14]

Summary

Introduction

The initiation phase of protein synthesis in eukaryotic cells is a complex series of highly regulated interactions between ribosomal subunits, mRNA, aminoacylated initiator methionyltRNA (Met-tRNAiMet), and eukaryotic translation initiation factors (eIFs).1 They all function to correctly position MettRNAiMet within the 80 S ribosomal P site at the correct AUG initiation codon of every mRNA [1]. We have determined the crystal structure of residues 544 –704 from yeast eIF2B⑀ at 2.3-Å resolution, and this fragment is an all-helical protein built around the conserved aromatic acidic (AA) boxes found in eIF4G and eIF5. An acidic belt and a second area in the C terminus containing residues from the AA boxes are important for binding to eIF2.

Results

Conclusion