PDCD4 inhibits translation initiation by binding to eIF4A using both its MA3 domains

Chikako Suzuki, Robert G. Garces, Katherine A. Edmonds, Sebastian Hiller, Sven G. Hyberts, Assen Marintchev*, and Gerhard Wagner*

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

Communicated by Charles C. Richardson, Harvard Medical School, Boston, MA, December 28, 2007 (received for review November 14, 2007) Programmed Cell Death 4 (PDCD4) is a protein known to bind MA3-c, MA3-m, and the eIF4G-MA3, also supporting a direct role eukaryotic initiation factor 4A (eIF4A), inhibit translation initiation, for the MA3-m domain in eIF4A binding and inhibition. Despite and act as a tumor suppressor. PDCD4 contains two C-terminal MA3 these observations, however, the exact function of MA3-m re- domains, which are thought to be responsible for its inhibitory mained unclear. Second, no structure was available for MA3-m. function. Here, we analyze the structures and inhibitory functions The sequence homology between MA3-m and MA3-c suggests a of these two PDCD4 MA3 domains by x-ray crystallography, NMR, similar fold but the extent of similarity was not clear. Furthermore, and surface plasmon resonance. We show that both MA3 domains it was open whether the two domains have similar or complemen- are structurally and functionally very similar and bind specifically tary function, and why PDCD4 needs two MA3 domains. to the eIF4A N-terminal domain (eIF4A-NTD) using similar binding Here, we report the crystal structure of the PDCD4 MA3-m interfaces. We found that the PDCD4 MA3 domains compete with domain and its NMR-derived eIF4A-binding face. We show that the eIF4G MA3 domain and RNA for eIF4A binding. Our data the PDCD4 MA3 domains compete with the eIF4G-MA3 and with provide evidence that PDCD4 inhibits translation initiation by RNA for binding to eIF4A. Both the structures of the two MA3 displacing eIF4G and RNA from eIF4A. The PDCD4 MA3 domains act domains and their eIF4A-binding faces are very similar. However, synergistically to form a tighter and more stable complex with the two PDCD4 MA3 domains act synergistically to form a tighter eIF4A, which explains the need for two tandem MA3 domains. and more stable complex with eIF4A, which explains the need for two tandem MA3 domains. apoptosis ͉ eIF4G ͉ protein ͉ NMR ͉ x-ray crystallography Results DCD4 is a tumor-suppressor protein that is up-regulated on MA3-m Is Structurally Similar to MA3-c but Contains an Additional Pinduction of apoptosis (1) and down-regulated in certain ag- C-Terminal Helix. A series of truncation mutations were carried out gressive tumors (2). PDCD4 is controlled by protein kinase S6K1 to identify the domain boundaries of human PDCD4 MA3-m. The and the ubiquitin ligase SCF␤TRCP, and its degradation is necessary region 157–302 (Fig. 1) exhibited the best NMR spectra and was for efficient protein translation in vivo, which is a prerequisite for thus assumed to contain the intact MA3-m domain. Backbone cell growth and, consequently, for cell proliferation (3). resonances for 142 of 158 nonprolines could be assigned by using PDCD4 is known to bind two eukaryotic translation initiation triple-resonance NMR experiments (reviewed in ref. 17). Similarly, factors eIF4A and eIF4G (4–6). eIF4A is an RNA helicase that 114 of 127 nonproline residues of human PDCD4 MA3-c were works as a subunit of eIF4F, a complex composed of eIF4G and assigned. A few short segments are invisible in the NMR spectra eIF4E. The helicase activity of eIF4A itself is weak but is enhanced presumably because of line broadening due to conformational upon binding to eIF4G (7, 8). eIF4G has two independent binding exchange. Chemical shift analysis indicates that MA3-m contains sites for eIF4A (9), one in the conserved middle domain (eIF4G-m, eight helices [supporting information (SI) Fig. 6A]. HEAT1/MIF4G) (Fig. 1), and the other in the adjacent second Both native and Se-Met MA3-m were crystallized. MA3-m HEAT domain (eIF4G-MA3, HEAT2/MA3) (reviewed in ref. 10). (Se-Met) crystals belonged to space group P212121 with cell NMR binding studies have shown that eIF4G-m interacts mainly axes a ϭ 37.68 Å, b ϭ 70.06 Å, c ϭ 110.88 Å, ␣ ϭ ␤ ϭ ␥ ϭ 90° with with the C-terminal domain of eIF4A (eIF4A-CTD) (11), whereas two monomers per asymmetric unit (SI Table 1). The structure was eIF4G-MA3 binds to the N-terminal domain of eIF4A (eIF4A- solved at 1.7 Å resolution and is shown in Fig. 1. The domain NTD) and only weakly to eIF4A-CTD (A.M., C.S., K.A.E., and consists of a stack of four ␣-helical hairpins, displaying a similar G.W., unpublished work). Mutation and deletion analysis indicates conformation as PDCD4 MA3-c (14) and eIF4G-MA3 (18). The that the interaction of eIF4A with eIF4G-m is necessary for crystal structure reveals that the folded part of MA3-m extends translation, whereas the interaction of eIF4A with eIF4G-c (eIF4G- between residues 161 (␣1) and 302 (␣8) and is Ϸ20 residues longer MA3ϩHEAT3 domain) plays a modulatory role (12). than previously predicted by analogy to MA3-c (14). The C- PDCD4 contains two MA3 domains after an N-terminal segment terminal 18 residues of the MA3-m fragment (after ␣8) have high of little known function (1). MA3 is a well conserved ␣-helical motif amino acid sequence homology to helix ␣9 in eIF4G-MA3. How- with typically 3–5 helical hairpins and is a subtype of HEAT domains. A single MA3 domain is found in eIF4G-c (eIF4G-MA3, Fig. 1), which has been reported to also bind eIF4A (10, 13). Author contributions: C.S., A.M., and G.W. designed research; C.S., R.G.G., and S.H. per- Recently, crystal and solution structures of mouse PDCD4’s C- formed research; C.S., R.G.G., K.A.E., S.H., S.G.H., and A.M. analyzed data; and C.S., R.G.G., terminal MA3 domain (MA3-c) were reported (14, 15). MA3-c was A.M., and G.W. wrote the paper. shown to bind eIF4A, compete with eIF4G-c, and was sufficient to The authors declare no conflict of interest. inhibit translation initiation. However, some questions remained. The atomic coordinates reported in this paper have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, www.pdb.org (PDB First, the function of the N-terminal MA3 domain (MA3-m) was ID code 2RG8). unclear. Mutations of conserved amino acid residues in either *To whom correspondence may be addressed at: Department of Biological Chemistry and MA3-c or MA3-m affect eIF4A binding, which implied that both Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA PDCD4 MA3 domains have eIF4A-binding abilities and contribute 02115. E-mail: gerhard࿝[email protected] or assen࿝[email protected]. in translation inhibition (16). NMR experiment revealed that This article contains supporting information online at www.pnas.org/cgi/content/full/ MA3-c binds to eIF4A-NTD through the loop between ␣5 and ␣6 0712235105/DC1. and the turn linking ␣3 and ␣4 (15), which is well conserved among © 2008 by The National Academy of Sciences of the USA

3274–3279 ͉ PNAS ͉ March 4, 2008 ͉ vol. 105 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712235105 Downloaded by guest on September 29, 2021 15 157 302 319 449 15N- MA3-m N- MA3-c A 105 human Pdcd4 1 MA3-m MA3-c 469 110 A B MA3 MA3 110 + 752 993 1235 1420 1438 1593 115 e I )mpp(N 115 human eIF4GI 1 eIF4G-m eIF4G-MA3 HEAT3 1600 F A4

HEAT1 HEAT2/MA3 HEAT3/W2 120 120 N- 51 T

eIF4G-c D253 D E249 E374 125 125 human eIF4AI 1 eIF4A-NTD eIF4A-CTD 406 L252 H421 T254 130 DEAD-box F176 N416 130 S422 105 B 110 1 2 3 C D MA3-m 157 -LPLDERAFEKTLTPIIQEYFEHGDTNEVAEMLRDLNLGEMKSGVPVLAVSLALEG-KAS 110 MA3-c 319 GGQQSVNHLVKEIDMLLKEYLLSGDISEAEHCLKELEVPHFHHELVYEAIIMVLESTGES + 115 ESTLER-SAI Ie F 4GMA3 1235 KAALSEEELEKKSKAIIEEYLHLNDMKEAVQCVQELASPSLLFIFVRHGV )m 115

4 5 6 pp A4 - MA3-m 215 HREMTSKLLSDL -CGTVMSTTDVEKSFDKLLKDLPELALDTPRAPQ