Previous studies have shown that the full-length Pdcd4, as well as a fragment of Pdcd4 containing the two MA3 domains alone, was capable of inhibiting eIF4A translation initiation activity and protein synthesis by binding to eIF4A. This fragment was capable of competing with the C-terminal domain of eIF4G (eIF4Gc) for binding to eIF4A (28
). The study of the cMA3 domain of Pdcd4, presented here, demonstrates how this domain alone mimics the structure and functional binding activity of eIF4Gc, leading to inhibition of translation activation activity of eIF4A.
The crystal structure of the cMA3 domain of tumor suppressor Pdcd4 and comparison with the structure of the MA3 domain of eIF4G have allowed us to characterize the structural features of the MA3 domain. First identified in Pdcd4 (a protein also known as MA3), these domains have also been found in DAP5 and nucleolar protein with MIF4G domain. Our structural analysis indicates that this domain displays significant structural homology to VHS domains and may be considered a subgroup of that family, as evidenced by the conservation of the number of pairs of repeated antiparallel helices and the angle of rotation between subsequent repeats. However, the MA3 domains of Pdcd4 and eIF4A are distinguished by the presence of an unusual turn at the end of helix 5. Interestingly, it is this helix that contains the residues shown to be important for the function of Pdcd4 and eIF4G in binding to eIF4A. Thus, we believe this helix to be an important eIF4A binding motif that distinguishes MA3 domains. While the manuscript was in preparation, an NMR secondary structure assignment was reported for the cMA3 domain of Pdcd4, and it agrees with our crystal structure data (27
Of the two MA3 domains found in Pdcd4, the cMA3 domain is more similar to the MA3 domain of eIF4G by sequence comparison. A comparison of the structures of the MA3 domains of eIF4Gc and Pcdc4 (cMA3) demonstrates a remarkable similarity between the structures as well. We also observed similarity in the positioning of conserved residues on one surface of the domains and in their electrostatic charge distribution properties. In particular, the residues Asp414 and Asp418, which are required for eIF4A binding in Pdcd4, are strictly conserved in eIF4G. In the N-terminal MA3 domain of Pdcd4, a glutamate residue replaces Asp414. In addition, Asp414 is replaced by a lysine residue in the MA3 domain of DAP5, which does not bind eIF4A (28
). These features suggested to us that the cMA3 domain of Pdcd4 may mimic the binding of eIF4Gc to eIF4A.
As predicted by this hypothesis, the Pdcd4 cMA3 domain alone functioned as an effective competitive inhibitor of the eIF4Gc MA3 domain. Interestingly, given the molar activity similar to that of Pdcd4 (0.8:1.0) and assuming similar protein stability, it appears that the cMA3 domain has affinity similar to, if not higher than, that of the eIF4Gc domain for eIF4A binding. Consistent with the idea that a molecule that competes for the eIF4Gc binding site on eIF4A may inhibit the function of eIF4A, the eIF4Gc domain was also shown to be sufficient to inhibit translation in vitro (20
). Although the mechanism of such inhibition is not clear, the eIF4A binding function of the cMA3 domain suggests that it may be through the disruption of the ideal stoichiometry of an active eIF4F complex. In this study, we show that eIF4Gc alone can inhibit stem-loop translation in vivo, and a similar result was seen for the Pdcd4 cMA3 domain. The relative importance of Pdcd4's competition for C-terminal function of eIF4G and its binding to the middle domain of eIF4G in translation inhibition has not been previously elucidated. The observation of similar efficiency for translation inhibition by cMA3, full-length Pdcd4, and eIF4Gc suggests that the competition with eIF4Gc for binding to eIF4A may play a major role in the mechanism of translation inhibition by Pdcd4.
We do not fully understand the function of the N-terminal MA3 domain in Pdcd4. It is expected to be structurally similar to the cMA3 domain structure presented here. However, attempts to build a three-dimensional homology model of the nMA3 domain structure based on the cMA3 domain structure were unreliable due to differences in residues that would likely result in differences in secondary structure and positioning of the helices relative to each other. Given the structural homology between the MA3 domain and the MIF4G domain, it is possible that Pdcd4 nMA3 might mimic the function of the MIF4G domain of eIF4G (eIF4Gm). A model built based on NMR interaction mapping data suggested that the MIF4G domain interacts with eIF4A and holds it in a closed and active conformation (22
). Although this may not be the case with Pdcd4, which functions to inhibit the eIF4A function, the nMA3 domain of Pdcd4 may compete with the eIF4G MIF4G domain for eIF4A binding. Another possibility is that the nMA3 domain mediates interactions observed between Pdcd4 and eIF4G. Further structural and functional studies of the nMA3 domain of Pdcd4 are required in order to fully understand the contributions of this domain to the inhibition of translation initiation and tumorigenesis by Pdcd4.
Phosphorylation of Pdcd4 by Akt (23
) appears to regulate Pdcd4 localization and certain activities. Since the Akt phosphorylation site (Ser457) is not part of the canonical MA3 structure of Pdcd4, it is not surprising that a mutation of this residue that renders it nonphosphorylated (Ser457Ala) also failed to affect competition for eIF4A binding. We have previously shown that mutations that abolish eIF4A binding (Asp414Lys and Asp418Ala) also inactivated translation inhibition by full-length Pdcd4 (29
). These results, along with our observation that mutations at the Akt site did not affect translation inhibition by Pdcd4 cMA3, agree with the conclusion that Akt phosphorylation does not influence cMA3 function. Furthermore, we observed that the mutation in the Akt site (S457) in the full-length protein also does not affect Pdcd4 function. Consistent with our observation, a recent report from Dorrello et al. (6
) supports our hypothesis that the other Akt site (S67) in the N terminus of the protein may play a significant role in the regulation of Pdcd4 expression and activity. In summary, our analyses indicate that although Akt phosphorylation may be important for posttranslational regulation of Pdcd4, phosphorylation at residue S457 clearly does not influence either binding of eIF4A to the cMA3 domain or the translation inhibition function of Pdcd4.
The translation initiation activity of eIF4A is important for mRNAs containing complex 5′ untranslated regions, many of which are involved in promoting oncogenesis. Given the ability of the cMA3 domain to inhibit this function on its own, this domain might form the basis for the design of drugs that target the translation of a subset of mRNAs to prevent or treat cancer.