(A) VWMD mutations tested in this study, visualized as spheres on the structure of S. pombe eIF2B (PDB: 5B04; Kashiwagi et al., 2016). The human mutation sites are shown in plain text and the corresponding yeast residues in superscript. For clarity, only one of each pair of residues is labeled. (B) Half-lives of GDP release for recombinant WT and VWMD eIF2B, calculated by fitting single-exponential decays to the Bodipy-FL-GDP fluorescence decay curves. All VWMD mutants except εR136H had reduced GEF activity that is significantly stimulated by ISRIB (p<0.001). Significance is shown for comparisons of each mutant to WT and between selected mutants. **p<0.005, ***p<0.001, ns, not significant. (C) ISRIB EC50 values calculated from dose-response measurements in GEF assays (individual curves shown in Figure 2—figure supplement 1A). Significance is shown for comparisons of each mutant to WT. **p<0.01, ***p<0.001. (D) Dose-response curves of GDP release half-life in the presence of increasing amounts of WT or V183F α subunit. For (B)-(D), half-lives of GDP release at each ISRIB/α concentration were calculated by fitting single-exponential decays to the Bodipy-FL-GDP fluorescence decay curves. Each point represents 9 measurements from three independent experiments (mean ±SD). (E) All eIF2B VWMD mutant complexes except εR136H have reduced complex stability compared to WT. Size-exclusion chromatograms of reconstituted wild-type and VWMD mutant eIF2B complexes in the presence of 300 mM NaCl. The elution positions of the (αβδγε)2 10mer and βδγε 4mer are indicated. A cartoon schematic of 10mer formation is shown above the graphs. (F) Quantification of eIF2B 10mer:4mer peak ratios from (E), which serves as a measure of complex stability (N = 3, mean ± SD). *p<0.05, ***p<0.001, ns, not significant. Significance is shown for comparisons to WT within a condition (vehicle or ISRIB). Between conditions for a given construct, all differences were significant (p<0.005).