Structure of the Catalytic Fragment of Translation Initiation Factor 2B and Identification of a Critically Important Catalytic Residue

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 eIF2B⑀ subunit. 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 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.

An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation

“…The potential interaction of the eIF5 GAP domain (NTD) (Das et al, 2001;Paulin et al, 2001) with eIF2g before the GAP activation on AUG recognition might help eIF2 retain the guanine nucleotide. In addition, the similarity of eIF5-CTD with eIF2Be-CTD (Boesen et al, 2004;Yamamoto et al, 2005;Wei et al, 2006) extends beyond the eIF2b interacting interface. The first two a-helices of eIF2Be-CTD are involved in catalysis and the domain interacts with eIF2g (Alone and Dever, 2006).…”

“…Based upon a homology modeled 3-D structure using the crystal structure of eIF2Be C-terminal HEAT domain (Boesen et al, 2004), several clusters of surface residues were independently mutated and their effects on interaction with MFC-binding partners and eIF4G were analyzed . The eIF5 alleles used and the interactions affected are summarized in Figure 4A and schematically in Figure 4B.…”

“…The regulatory subcomplex binds eIF2a (Krishnamoorthy et al, 2001), whereas the catalytic subcomplex binds eIF2b and stimulates release of the guanine nucleotide bound to eIF2g (Gomez et al, 2002). Of the catalytic subcomplex, the C-terminal domain (CTD) of eIF2Be (residues 518-712) is folded into a HEAT domain composed of eight a-helices, with the GEF catalytic center at the first two a-helices and a strong eIF2b-binding site at the last three a-helices (Boesen et al, 2004). This conserved eIF2b-binding motif within eIF2Be has been termed aromatic/acidic (AA-) boxes 1 and 2 and interacts with the lysine-rich boxes (K-boxes) found in the eIF2b N-terminal domain (NTD) (Asano et al, 1999).…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Though neither GDI mutation significantly alters growth of yeast on rich or minimal medium, they dramatically impair responses to eIF2 phosphorylation, indicating an important role of eIF5 in tight regulation of eIF2B GEF activity. eIF2B displaces eIF5 from eIF2-GDP: Because the CTD of eIF5 and the GEF domain of eIF2Be (see Figure 3, C and D) share a common HEAT repeat structure (Boesen et al 2004;Bieniossek et al 2006) necessary for binding to eIF2b (Asano et al 1999), binding of each factor to eIF2 is mutually exclusive (Jennings and Pavitt 2010b). eIF5 must dissociate from the stable eIF2-GDP/eIF5 complex to enable eIF2B GEF action; however, eIF2B itself can displace eIF5 (Jennings et al 2013).…”

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

“…A third subunit, eEF1Bb (formerly EF-1d), is found only in metazoans and exhibits catalytic GEF activity, although its role in the cell is not well understood (van Damme et al 1990). The minimal catalytic fragments of eIF2Be and eEF1Ba show no conservation in sequence or structure (Andersen et al 2000;Boesen et al 2004).…”