1. Volume 48, Issue 3, Pages 375-386 (November 2012)
Interdomain Allostery Promotes Assembly of the Poly(A) mRNA Complex with PABP and eIF4G 
Nozhat Safaee, Guennadi Kozlov, Anne M. Noronha, Jingwei Xie, Christopher J. Wilds, Kalle Gehring 
Molecular Cell 
Volume 48, Issue 3, Pages (November 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 48, 375-386DOI: (10.1016/j.molcel.2012.09.001)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 Protein-Protein Interactions of eIF4G and PABP
(A) Binding sites on human eIF4GI for various translation factors. The sequence of the fragment of eIF4G used in our crystallization studies is shown. The binding site for PABP overlaps with the one for NSP3 of rotaviruses.
(B) Domains of human PABP and binding sites. The sequence of the four RRM domains is shown along with the secondary structure. BLUSOM62 score was used to map the conserved residues. Asterisks indicate the positions of residues that are important for RRM2 binding to eIF4G.
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4. Figure 2 Residues 178–203 of eIF4G Bind to the RRM2 Domain of PABP
(A) 1H,15N HSQC spectra of 15N-RRM2 (PABP residues 86–192) in the absence (black) and presence (orange) of Nt4G at an RRM2:Nt4G molar ratio of 1:2. Unlike RRM2, RRM1 does not interact with eIF4G (see Figure S1).
(B) Weighted average of Nt4G-induced chemical shift changes in RRM2 plotted by residue number. Degree symbols indicate residues (97, 103, 105, 120, 121, 134, 137, 140, 158, 160, 167, 168) whose signals disappeared in the complex and were assigned a shift change of 0.15 ppm. Asterisks denote residues that were not assigned.
(C) Nt4G-induced chemical shift changes (orange) mapped onto the RRM2 crystal structure. Spectral changes occurred throughout RRM2 with the largest changes in helix α1 and in the loop between strands β4 and β5.
(D and E) HSQC spectra of 15N-Nt4G alone and in the presence of RRM2 in a Nt4G: RRM2 molar ratio of 1:3.
(F) Weighted average of RRM2-induced chemical shift changes in Nt4G plotted by residue and the secondary structure observed in the crystal structure. Asterisks indicate residues (178–179, 192–193) that were only assigned in the spectrum of the RRM2-bound form of Nt4G. Degree symbols indicate residues (181–183, 188, 190) whose signals disappeared in the complex and were assigned a shift change of 0.8 ppm. The spectra identify residues 178–203 as the region of eIF4G that binds RRM2.
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5. Figure 3
Crystal Structure of the Ternary Complex of Poly(A)·PABP·eIF4G
(A) Structure of the ternary complex of eIF4G ( ) (brown) bound to PABP RRM1-2 (light blue) in the presence of poly(A)11 (dark blue). The eIF4G peptide binds to the α-helical surface of RRM2, while the poly(A) RNA binds to the β sheet surface of both RRMs.
(B) Hydrophobic interactions between eIF4G and RRM2. eIF4G I180 plays a key role and penetrates into the hydrophobic core of RRM2. Additional surface contacts are made by I182 and the eIF4G C-terminal α helix. eIF4G residue numbers are italicized to distinguish them from PABP.
(C) Polar contacts (dashed lines) contribute to eIF4G and RRM2 binding. Backbone hydrogen bonds between the first β strand of eIF4G (residues R181–R183) and the fourth β strand of RRM2 (residues 162–164) mimic formation of an extended sheet structure. The side chains of N164 and D165 of PABP make additional polar contacts to R181 and R183 of eIF4G (data not shown).
(D and E) Helices following RRM1 (residues 92–96) and RRM2 (residues 176–183) contribute to RNA binding. R94 makes hydrogen bonds with the hydroxyl oxygen (O2′) of the ribose moieties rA5 and rA6 while R179 forms hydrogen bonds with the phosphate oxygens of rA2.
For the details of crystal packing and comparisons with related structures, see Figure S2.
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6. Figure 4 Cooperative Binding of eIF4G and Poly(A) to PABP
(A) Gel shift assay of 32P-labeled poly(A)11 RNA binding to RRM1-2 and Nt4G. In two series of six reactions, addition of RRM1-2 leads to formation of the binary complex of RRM1-2·poly(A)11 and retards the electrophoretic mobility of the 32P-labeled RNA. In the presence of GST-Nt4G, an additional band appears due to formation of the ternary complex of poly(A)11·RRM1-2·GST-Nt4G. The control lane “+ cold RNA” contains excess unlabeled poly(A)11 which competes for binding to RRM1-2. The control lane “+ Nt4G” shows that poly(A) does not interact with GST-Nt4G in the absence of RRM1-2. Quantification (right panel) of the bands corresponding to unbound 32P-labeled poly(A)11 allows estimation of the affinity constants of RNA binding to RRM1-2 in the absence and presence of GST-Nt4G.
(B) Gel shift assay of 32P-labeled poly(A)25 RNA binding to RRM and Nt4G. Addition of RRM leads to formation of the binary complex of RRM ·poly(A)25. In the presence of GST-Nt4G, a more slowly migrating band appears due to formation of the ternary complex of poly(A)25·RRM ·GST-Nt4G. Quantification (right panel) of the binary (no addition) and ternary complex formation (+Nt4G) confirms the higher affinity of the ternary complex.
(C) ITC data for RRM1-2 binding poly(A)11 to form the binary complex, ΔH = −32,590 ± 181 cal/mol, Kd = 0.6 ± 0.03 μM, N = 1.04 (left panel) and RRM1-2·Nt4G binding poly(A)11 to form the ternary complex, ΔH = −36,352 ± 189 cal/mol, Kd = 62 ± 8.4 nM, N = 0.98 (right panel).
(D) ITC data for RRM1-2 binding Nt4G to form the binary complex, ΔH = −8,844 ± 147 cal/mol, Kd = 19.7 ± 1.5 μM, N = 1.06 ± 0.01 (left panel) and RRM1-2·poly(A)11 binding Nt4G to form the ternary complex, ΔH = −12,790 ± 111 cal/mol, Kd = 1.6 ± 0.1 μM, N = 1.00 (right panel).
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7. Figure 5
Solution Conformations of the RRM1-2-3 of PABP and Its Effect on eIF4G Binding
(A) The ab initio bead models of the free RRM1-2-3 fragment (blue) and RRM1-2-3·poly(A)11 complex (green) with their corresponding maximum diameters (Dmax) as calculated by GNOM. Each model represents the averaged shape obtained from 50 models calculated by DAMMIF and averaged by DAMAVER. The overlay of the two shapes shows the change in the conformation upon poly(A) binding. For comparison, the crystal structure of the isolated RRM2 domain (gray) is shown.
(B) The pair distribution plots of RRM1-2-3 alone and in complex with poly(A)11 modeled by GNOM. The calculated Rg of the RRM1-2-3 fragment free and in complex with poly(A) were 30.8 ± 0.5 Å and 37.6 ± 0.4 Å, respectively.
(C) ITC data for RRM2 binding Nt4G, ΔH = −24,370 ± 430 cal/mol, Kd = 5.3 ± 0.03 μM, N = 1.03 ± 0.01 (left panel), RRM1-2 binding Nt4G, the same as in Figure 3C, left panel shown for the sake of comparison (middle panel) and RRM2-3 binding Nt4G, ΔH = −11,310 ± 202 cal/mol, Kd = 5.9 ± 0.03 μM, N = 1.02 ± 0.01 (right panel).
See also Figure S3.
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8. Figure 6
Removal of RNA Decreases the Interaction between Endogenous PABP and eIF4G
Immunoprecipitation (IP) assay of HeLa cell extracts untreated or treated with thapsigargin (TG).
(A) The cells were either untreated or treated with TG for 1 hr prior to harvest. The precleared cell extracts were probed for eIF2α and phosphorylated eIF2α (P-eIF2α). The higher amount of P-eIF2α confirms the successful treatment with TG.
(B) The IP reactions were carried out on the cell extracts from both the untreated and TG-treated cells. The cell extracts were either untreated or treated with nuclease (Cyanase) or GST-Paip2 (22-75), where indicated. The amount of eIF4G bound to PABP was analyzed in western blots using α-eIF4GI antibody.
(C) Quantification of the amount of eIF4GI bound to PABP in the IP reactions. The amount of bound eIF4GI was divided by the amount of immunoprecipitated PABP and the values normalized relative to the untreated extract.
(D) Analysis of the total HeLa cell lysates (TCL) and the depleted lysates corresponding to each IP experiment, using antibodies against eIF4GI, PABP, and β-actin in western blot assay. Bottom panel shows the nucleic acid content of the untreated and nuclease-treated samples using agarose gel electrophoresis (0.8% gel) in 1 × TAE buffer, detected by ethidium bromide (EtBr), which confirms the removal of nucleic acid content of the lysates by nuclease treatment.
Molecular Cell  , DOI: ( /j.molcel )
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