Fig. 5. Nelfinavir treatment inactivates ribosomes via eEF2.
A Representative polysome profiles following treatment with vehicle (black) or nelfinavir (red). F11 cells were treated with either vehicle (Veh) or nelfinavir (NFV) for 1 h, followed by 5 min of cycloheximide (CHX, 100 µg/ml). Cells were lysed and used to generate polysome profiles. B Representative immunoblots from three biological replicates of ribosomes purified by sucrose cushion (cropped to depict one representative band per condition). F11 cells were treated with either vehicle (Veh) or nelfinavir (NFV) for 1 h, followed 100 µM emetine for 5 min. Cells were lysed and loaded on 30% sucrose cushions before ultracentrifugation to pellet ribosomes. An additional vehicle sample was further treated with EDTA (30 µM) to dissociate polysomes prior to loading on sucrose cushion. Immunoblots were performed using input and resuspended ribosome pellets. C Cryo-EM structure of eEF2-bound 80S mouse ribosome in the rotated state. The large subunit (LSU) is shown in pale cyan, the small subunit in pale yellow (SSU), eEF2 in marine, SERBP1 in purple, and E-site tRNA in orange. D Overlay of eEF2 (marine) and p-eEF2 (purple) structures shows nearly identical conformations (RMSD 0.256 Å2 for corresponding Calpha atoms). The two structures differ in the presence of an ordered switch I in the unphosphorylated eEF2 (marine) near the bound GDP. E Density of eEF2 (marine) and p-eEF2 shows the presence of switch I in the unphosphorylated eEF2 structure. F eEF2 Thr56, target of eEF2K, is surrounded by negatively charged residues Asp57, Asp36, and Asp103 and is oriented towards the GDP beta-phosphate. This suggests that switch I rearranges upon Thr56-phosphorylation due to electrostatic repulsion. Uncropped blots are presented in Supplemental Fig. 7.