Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions

Abstract

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5' end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-A resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.

Fig. 1.

Domain organization of eIF4G and eIF4A. (A) Schematic drawing of the domain organization of eIF4G and its binding sites. The scale bar indicates the amino acid positions. (B) Schematic drawing of eIF4A. The conserved sequence motifs of the DEAD-box helicase family are color-coded as follows: Q, magenta; Walker A/I, blue; IA, cyan; IB, green; Walker B/II, yellow; III, orange; IV, brown; V, red; and VI, magenta. The scale bar is very approximate; the N-terminal domain spans residues 1–221, the C-terminal domain residues 225–391.

Fig. 2.

Pull-down experiments using His-tagged yeast eIF4A in conjunction with yeast eIF4GI. His-tagged full-length eIF4A (A) or eIF4A-CTD (B) was bound to a Ni-NTA column. eiF4G proteins (10× excess) were added and incubated for 5 min. After washing out unbound proteins with 3 × 10 volumes of buffer, bound proteins were specifically eluted with 100 mM imidazole, separated on 15% SDS gels, and visualized by Coomassie staining. (A) Binding to full-length eIF4A. Markers: 1, eIF4G (572–853) wash; 2, eIF4G (572–853) elution; 3, eIF4G (572–853)(W579A) wash; 4, eIF4G (572–853)(W579A) elution; 5, eIF4G (592–862) wash; 6, eIF4G (592–862) elution; 7, 8, and 9, unspecific (background) adsorption of eIF4G proteins added to Ni-NTA columns in the absence of eIF4A. (B) Binding to eIF4A-CTD. Markers: 1, eIF4G (572–853) wash; 2, eIF4G (572–853) elution; 3, eIF4G (572–853)(W579A) wash; and 4, eIF4G (572–853)(W579A) elution.

Fig. 3.

Stimulation of eIF4A's ATPase activity by various eIF4G proteins. eIF4A was incubated in the absence or presence of 2.5-fold excess eIF4G protein and ATP and RNA at 22°C by using EnzChek phosphate assay. Phosphate release was measured for 15min. (A) Activity of yeast eIF4A (wild-type and mutant). 1, eIF4A + buffer (negative control); 2, eIF4A + eIF4G (572–853); 3, eIF4A(R35D) + buffer; 4, eIF4A(R35D) + eIF4G (572–853); 5, eIF4A + eIF4G (572–853)(D842R); 6, eIF4A + eIF4G (592–862); 7, eIF4A + eIF4G (572–853)(W579A); and 8, eIF4A + human eIF4G(710–1056). (B) Activity of human eIF4A in the presence of: 1, buffer; 2, human eIF4GI(710–1056); and 3, human eIF4GI (710–970).

Fig. 4.

Overall structure of the eIF4G–eIF4A complex. (A) Ribbon diagram of the x-ray structure of the complex. eIF4A is shown at the bottom in dark gray and light gray for the N-terminal and C-terminal domain, respectively. DEAD-box motifs are color-coded as follows: Q, magenta; Walker A/I, blue; IA, cyan; IB, green; Walker B/II, yellow; III, orange; IV, brown; V, red; and VI, magenta. AMP is shown as yellow sticks. eIF4G (572–853) is shown at the top and is colored in light blue; motifs M1–M4 are shown in tan. Some key residues mentioned in the text are shown as sticks. The dashed loop from eIF4G residues 582 to 600 (Left, blue) is modeled. (B) View rotated around the vertical by ≈180°. The salt bridge between eIF4A-NTD Arg-35 and eIF4G Asp-842 is indicated (Upper Left).

Fig. 5.

Characterization of the W579A mutant. (A) Electron density around Trp-579. eIF4G C-atoms are cyan; eIF4A carbon atoms are green. Residues 577–582 were omitted from the phase calculation. The 2F_o-F_c map is contoured at 1.0 standard deviations above the mean. (B) Temperature-sensitive phenotype of the tif4631 W579A mutant. In vivo selection for yeast clones carrying the full-length W579A mutation was performed as described in Materials and Methods. The upper two sectors correspond to wild-type cells, the lower sector shows growth of the W579A mutant at 30°C (Left) and 37°C (Right).