Extended Data Fig. 4: Comparison of eIF2B HDX-MS protection by NSs and 2BAct.
From: A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling
a,b, Representative percent deuteration difference map with overlaid time points for (a) apo versus NSs-bound eIF2B(αβγδε)2 decamers and (b) apo versus 2BAct-bound eIF2B(αβγδε)2 decamers. Positive values represent peptides that exchanged less deuterons in the 2BAct—bound state. Solid colored bars indicate each eIF2B subunit, corresponding to color scheme in Fig. 1. On each graph, timepoints are overlayed and color-coded: 10s deuteron difference mapped in red, 100s difference mapped in orange, 15 min difference mapped in cyan, 3 hr difference mapped in dark blue. Positive values represent peptides with more protection (less deuteration) in the second listed state relative to the first listed state. Significant protection is defined as multiple peptides with ∆#D greater than 0.5 deuterons; peptides without significant protection are contained within the dimmed threshold. Regions of NSs protection located at eIF2Bα effector binding site are shown in orange and purple boxes. The 2BAct effector binding site is indicated by pink boxes. The eIF2Bδ C-terminal ‘Switch-Helix’ is indicated by green boxes. (c) apo versus 2BAct-bound eIF2B(αβγδε)2 decamers and apo versus NSs-bound eIF2B(αβγδε)2 decamers difference maps plotted onto the same ∆#D axes. Timepoints are overlaid and color-coded; NSs 15 min is mapped in black, NSs 3 hr is grey, 2BAct 15 min is cyan, and 2BAct 3 hr is dark blue. Regions of NSs protection located at eIF2Bα effector binding site are shown in orange and purple numbered boxes. The eIF2Bδ C-terminal ‘Switch-Helix’ is indicated by green numbered box. Regions specifically protected by 2BAct indicated with pink numbered boxes; regions protected by both 2BAct and NSs are indicated with yellow numbered boxes. (d) Numbered regions from (c) are mapped onto eIF2B-NSs structure (PDB 7RLO) with 2BAct binding site shown. (e) Representative peptide uptake plots comparing 2BAct-bound (blue) vs NSs-bound (grey) vs apo eIF2B (black). The total number of exchanged deuterons per condition are plotted over time (n = three independent experiments) ± SD not corrected for back exchange.
