Abstract
One of the key regulatory points of translation initiation is recruitment of the 43S preinitation complex to the 5′ mRNA cap by the eIF4F complex (eIF4A, eIF4E, and eIF4G). The tumor suppressor protein Pdcd4 has been shown to inhibit cap-dependent translation by interacting tightly with the RNA helicase eIF4A via its tandem MA-3 domains. The NMR studies reported here reveal a fairly extensive and well defined interface between the two MA-3 domains in solution, which appears to be stabilized by a network of interdomain salt bridges and hydrogen bonds, and reveals a unique orientation of the two domains. Characterization of the stoichiometry of the Pdcd4-eIF4A complex suggests that under physiological conditions Pdcd4 binds to a single molecule of eIF4A, which involves contacts with both Pdcd4 MA-3 domains. We also show that contacts mediated by a conserved acidic patch on the middle MA-3 domain of Pdcd4 are essential for forming a tight complex with eIF4A in vivo, whereas the equivalent region of the C-terminal MA-3 domain appears to have no role in complex formation in vivo. The formation of a 1:1 eIF4A-Pdcd4 complex in solution is consistent with the reported presence in vivo of only one molecule of eIF4A in the eIF4F complex. Pdcd4 has also been reported to interact directly with the middle region of eIF4G, however, we were unable to obtain any evidence for even a weak, transient direct interaction.
Highlights
The highly conserved eukaryotic protein Pdcd43 was initially discovered in a screen for genes activated during apoptosis [1] and subsequently identified as a tumor suppressor in studies of a mouse keratinocyte model of tumor promotion [2]
We show that contacts mediated by a conserved acidic patch on the middle MA-3 domain of Pdcd4 are essential for forming a tight complex with eukaryotic translation initiation factor 4A (eIF4A) in vivo, whereas the equivalent region of the C-terminal MA-3 domain appears to have no role in complex formation in vivo
Mapping of the Pdcd4 MA-3M/MA-3C Intra-molecular Interface—The positions of NMR signals from the backbone carbonyl and amide groups in proteins are highly sensitive to changes in their local environment, and shifts in these signals were used to localize the intra-molecular interface between the Pdcd4 MA-3 domains
Summary
The highly conserved eukaryotic protein Pdcd (programmed cell death protein 4) was initially discovered in a screen for genes activated during apoptosis [1] and subsequently identified as a tumor suppressor in studies of a mouse keratinocyte model of tumor promotion [2]. Pdcd has been shown to compete with RNA and the eIF4G MA-3 domain for binding to eIF4A, with complex formation resulting in the inhibition of cap-dependent translation [14, 15, 21, 22, 38]. Pdcd has been reported to interact with eIF4Gm in both the presence and absence of eIF4A [14, 39] The structures of both the isolated C-terminal (MA-3C) and middle (MA-3M) MA-3 domains of Pdcd have been solved and are shown to be composed of three and four layers of atypical HEAT repeats, respectively, as shown, c and f [20, 22, 38]. The complex structures revealed two molecules of eIF4A bound to a single molecule of Pdcd via distinct interaction modes
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