Figures and data
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_tbl1.tif/full/max/0/default.jpg)
Performance of the PPI-HotspotID vs. FTMap and SPOTONEa
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_tbl2.tif/full/max/0/default.jpg)
Performance of AlphaFold-Multimer, PPI-HotspotID, and their combination for 48 “unsolved” complex structures
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig1.tif/full/max/0/default.jpg)
(Top left) The X-ray structure (PDB: 1IAR) of interleukin-4 (grey) in complex with interleukin-4 receptor subunit alpha (wheat) with five PPI-hot spots; interface PPI-hot spots (E13, R92, and N93) are in blue and the non-interface ones (K127 and Y128) are in green. The PPI-hot spot numberings are based on the interleukin-4 free structure (PDB: 1BBN). The correct predictions of PPI-hotspotID (top right), FTMap (bottom left) and SPOTONE (bottom right) are mapped to the corresponding residues of the complex structure.
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig2.tif/full/max/0/default.jpg)
Evaluation of the predicted CPEB2-interacting amino acid residues in eEF2. (a) Salient features of mouse eEF2, showing the various domains and the mutated amino acids in domain V. mut 1, G778A, T779A, and P780A; mut 2, F794A; mut 3, D815A. (b) Reciprocal co-IP. The 293T cells expressing myc-CPEB2 along with wt or mutant flag-eEF2 or control GFP were harvested and then precipitated with flag or myc IgG. The precipitated substances were used for western blotting with myc and flag antibodies. IP, immunoprecipitation; IB, immunoblotting; IgG H.C., IgG heavy chain. (c) HeLa cells transfected with the plasmid expressing shRNA against human eEF2 (siheEF2) were harvested after 4-day puromycin selection for western blotting. HeLa cells transfected with the eEF2 knockdown plasmid along with flag-tagged wt or mutant mouse eEF2 after 4-day selection with puromycin and G418 were used for (d) S35-met/cys-labeling of synthesized proteins or (e) western blotting with the denoted antibodies. The normalized HIF-1α protein level (HIF-lα/β-actin signal) was calculated and expressed as mean ± SEM from three independent experiments. 2-tailed Student’s t-test, * < 0.05.
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig3.tif/full/max/0/default.jpg)
(Top) The structure (PDB 2N73)83 of GCP60 (green) in complex with PI4K-b (cyan) with the GCP60-PI4K-b interface encircled. (Bottom) The four experimentally known PPI-hot spots of GCP60 are shown in red. H45 and Y49 form hydrogen bonds across the interface with PI4K-b. Although F19 and Y46 do not directly contact PI4K-b, F19 is in vdW contact with Q42, which in turn forms vdW contacts with H45, whereas Y46 is in vdW contact with both H45 and Y49.
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig4.tif/full/max/0/default.jpg)
(Left) The structure (PDB 2OF5)67 of 7 CRADD proteins in complex with 5 PIDD proteins. The circle shows the CRADD-CRADD interface between chains C (cyan) and G (orange), whereas the other 5 CRADD chains are in gray, and the 5 PIDD proteins are in green. (Right) G128 (red) in CRADD (chain C) participates indirectly in CRADD-CRADD interactions via a backbone–side chain hydrogen bond between its neighbor, L127, and R147 in another CRADD (chain G).
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig5.tif/full/max/0/default.jpg)
Uncropped immunoblot images.
The uncropped images for Figures 2b, 2c, and 2e are shown with indicated molecular weight marker.
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig6.tif/full/max/0/default.jpg)
Uncropped immunoblot images of the entire membranes for Figure 2b.
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig7.tif/full/max/0/default.jpg)
Uncropped images of the entire membrane for Figure 2c and phosphoimager file for 2d.
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig8.tif/full/max/0/default.jpg)
Uncropped immunoblot images of the cut membranes for Figure 2e.
![](https://prod--epp.elifesciences.org/iiif/2/96643%2Fv1%2F3400169%2F3400169v2_fig9.tif/full/max/0/default.jpg)
Primer sequences.
The sequences of sense (forward) and antisense (reverse) primers used for site-directed mutagenesis.