Crystal structure of the eIF4A-PDCD4 complex

Abstract

Tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5'-untranslated region of mRNAs and controls the initiation of translation. Here, we determined the crystal structure of the human eIF4A and PDCD4 complex. The structure reveals that one molecule of PDCD4 binds to the two eIF4A molecules through the two different binding modes. While the two MA3 domains of PDCD4 bind to one eIF4A molecule, the C-terminal MA3 domain alone of the same PDCD4 also interacts with another eIF4A molecule. The eIF4A-PDCD4 complex structure suggests that the MA3 domain(s) of PDCD4 binds perpendicular to the interface of the two domains of eIF4A, preventing the domain closure of eIF4A and blocking the binding of RNA to eIF4A, both of which are required events in the function of eIF4A helicase. The structure, together with biochemical analyses, reveals insights into the inhibition mechanism of eIF4A by PDCD4 and provides a framework for designing chemicals that target eIF4A.

Fig. 1.

PDCD4-mediated eIF4A inhibition models. (A) Overall structure of the 2:1 eIF4A–PDCD4 complex. Each eIF4A structure is formed with two α/β domains (NTD and CTD) and colored in blue and green. The PDCD4 is formed with mMA3 (orange) and cMA3 (magenta) domains. (B) The mMA3 (orange) and cMA3 (magenta) of PDCD4 bind to the interface between the NTD and CTD of eIF4A (blue) in the two-MA3-binding mode. The interacting secondary structures are labeled. (C) eIF4A–PDCD4 complex in the cMA3-binding mode. The eIF4A molecule is in the same orientation as in Fig. B. Here, cMA3 (magenta) is positioned in the same eIF4A-binding site of mMA3 of PDCD4 in the two-MA3-binding mode. mMA3 (orange) is directed toward the CTD of eIF4A, and only one hydrogen bond is observed between mMA3 (Ser-393) and the CTD (Met-216). (D) Surface representation of the PDCD4 structure in two different views. The eIF4A-binding region in the two-MA3- and cMA3-binding mode is colored in blue and magenta, respectively. (E) Surface representation of the two eIF4A structures. The PDCD4-binding site in the two-MA3-binding mode is colored orange (Left) and in the cMA3-binding mode is colored magenta (Right).

Fig. 2.

Structural analyses of the eIF4A–PDCD4 interface. (A) mMA3 (orange) and NTD of eIF4A (light blue) interact primarily through ion pairs and hydrogen bonds. In addition to the interactions described in the text, an ion pair between Lys-212 of mMA3 and Glu-186 of NTD further stabilizes the interactions between the mMA3–NTD surface groove. Ion pairs and hydrogen bonds are represented by dots. Oxygen and nitrogen atoms are shown in red and blue, respectively. (B) cMA3 (magenta) and NTD of eIF4A (green) interact primarily through ion pairs and hydrogen bonds. Helices 15 and 16 and intervening loops from cMA3 bind to the surface groove formed by helices 4, 5, 6, and 7 in NTD. Ion pairs and hydrogen bonds are represented by dots. Oxygen and nitrogen atoms are shown in red and blue, respectively. (C) Helices 1 and 2 from mMA3 (orange) bind to the shallow groove formed by helices 11, 12, and 14 in the CTD (light blue), forming another eIF4A–PDCD4 interface. (D) Helices 9 and 10 from cMA3 (magenta) bind to the shallow groove formed by helices 11 and 14 in CTD (green), forming another interface. In addition to the interactions described in the text, Glu-330 and Glu-337 from cMA3 form an ion pair network with Arg-282 from CTD. (E) Interface between the cMA3 (pink) and CTD of eIF4A (green). In addition to a hydrophobic cluster at the center of the interface, five hydrogen bonds and two ion pairs further stabilize this interface. (F) Overall structure of the eIF4A–PDCD4 complex in the same view of A–E.

Fig. 3.

Inhibitory activities of PDCD4 mutant proteins. (A) (Left) SEC analysis of the eIF4AΔN–PDCD4ΔN (class 1) mutant proteins. The quadruple mutant contains four mutated residues, E249A, E253A, D414A, and D418A. Before SEC analysis, the WT (blue line, 50 μM) or mutant PDCD4ΔN proteins and eIF4AΔN were incubated at 4 °C for 1 h in a 1:1 molar ratio. WT2 (dotted line) represents the 2:1 eIF4AΔN–WTPDCD4ΔN complex. Although the WT2 complex shows no unbound eIF4A or PDCD4ΔN, the WT complex exhibits notable amount unbound PDCD4. However, a 1:1 mixture of eIF4AΔN–PDCD4ΔN mutant proteins did not show much of the free PDCD4ΔN. The standard molecular masses were the same as those in Fig. S1D. (Right) SEC analysis of the eIF4AΔN–PDCD4ΔN (class 2) mutant proteins. (B) Inhibition of eIF4A helicase activities by various PDCD4ΔN mutant proteins. The unwinding effects of 13-bp dsRNA by eIF4A in the absence and presence of various PDCD4ΔN mutant proteins are shown. Total amount of unwound product was quantified by a PhosphorImager and expressed as a percentage of total radiolabel (Bottom). −4AΔN/−PDCD4ΔN and Δ represent reaction mixtures in the absence of eIF4A at 0 °C and 95 °C, respectively. Each reaction mixture contains 2 nM dsRNA, 3 μM full eIF4A, and 6 μM various PDCD4ΔN mutant proteins. Inhibition of eIF4A by an excess amount of class 2 PDCD4 mutant proteins is shown in Fig. S7A. (C) In vitro translation inhibition by various PDCD4ΔN mutant proteins. The percentage of translation occurring in the absence and presence of each mutant is indicated. The luciferase activity in the absence of WT or mutant PDCD4ΔN proteins was designated as 100%. Different concentrations of various PDCD4ΔN mutants were used: orange bars, no PDCD4ΔN; yellow bars, 2 μM; blue bars, 8 μM; green bars, 72 μM. Results indicate the means ± SEM from triplicate experiments.

Fig. 4.

Mechanism of the eIF4A inhibition by PDCD4. (A) Structural superposition of the closed form of Vasa (green) that bound to dsRNA (space-filling model, oxygen in red, nitrogen in blue, and carbon in yellow) and the relatively open form of eIF4A (light blue) that bound to cMA3 of PDCD4 (magenta) in the one-MA3 inhibition model. The cMA3 domain masks the dsRNA-binding site in eIF4A. The ATP analog is indicated by a thick line (dark blue). Conformational change of PDCD4-bound eIF4A to the closed form of Vasa is shown with an arrow. (B) Schematic diagram of the PDCD4-mediated inhibition of eIF4A. The domain closure movement of eIF4A (blue) induced by ATP (small red circle) to unwind dsRNA (yellow) is blocked by the binding of the two MA3 domains (orange and magenta) of PDCD4 to the interface of eIF4A.