Identification of Intersubunit Domain Interactions within Eukaryotic Initiation Factor (eIF) 2B, the Nucleotide Exchange Factor for Translation Initiation

In eukaryotic translation initiation, eIF2B is the guanine nucleotide exchange factor (GEF) required for reactivation of the G protein eIF2 between rounds of protein synthesis initiation. eIF2B is unusually complex with five subunits (alpha-epsilon) necessary for GEF activity and its control by phosphorylation of eIF2 alpha. In addition, inherited mutations in eIF2B cause a fatal leukoencephalopathy. Here we describe experiments examining domains of eIF2B gamma and epsilon that both share sequence and predicted tertiary structure similarity with a family of phosphohexose sugar nucleotide pyrophosphorylases. Firstly, using a genetic approach, we find no evidence to support a significant role for a potential nucleotide-binding region within the pyrophosphorylase-like domain (PLD) of eIF2B epsilon for nucleotide exchange. These findings are at odds with one mechanism for nucleotide exchange proposed previously. By using a series of constructs and a co-expression and precipitation strategy, we find that the eIF2B epsilon and -gamma PLDs and a shared second domain predicted to form a left-handed beta helix are all critical for inter-protein interactions between eIF2B subunits necessary for eIF2B complex formation. We have identified extensive interactions between the PLDs and left-handed beta helix domains that form the eIF2B gamma epsilon subcomplex and propose a model for domain interactions between eIF2B subunits.