Figure 5.
ISRIB Inhibits Binding of eIF2B to the N-Terminal Domain of Phosphorylated eIF2α
(A) Biolayer interferometry (BLI) traces of the association and dissociation phases of eIF2B decamers (100 nM) or eIF2Bβδγε tetramers (400 nM) in the absence (DMSO, in blue) or presence of ISRIB (1 μM, in pink) to and from the biotinylated P-eIF2α-NTD immobilized on the BLI probe. The fits to a 2-phase association and a 2-phase dissociation model are indicated by the gray dashed line. Shown are mean ± SD (n = 3) of the sample of eIF2B decamers with and without ISRIB and all the data points of the tetramer samples from a representative experiment conducted three times.
(B) Left: BLI traces of consecutive association phases and a terminal dissociation phase of wild-type and the indicated eIF2B mutants in the absence (DMSO, in blue) or presence of ISRIB (1 μM, in pink) as in (A). The probe was reacted with escalating concentrations of eIF2B (9–150 nM) ± ISRIB, before dissociation in the respective buffer. Shown are all the data points of a representative experiment performed three times. Right: plots of the mean and 95% confidence interval (CI) of the plateau values of the association phases (obtained by fitting the data from the traces on the left to a 2-phase association non-linear regression model) against the concentration of eIF2B. The dotted line reports on the fit of plots to a one site specific binding with Hill slope = 1 non-linear regression model.
(C) Left: time-dependent change in BLI signal in the dissociation phase of eIF2B (previously associated in absence of ISRIB) from the biotinylated P-eIF2α-NTD in the presence of escalating concentration of ISRIB. The mean and 95% CI of the fraction of the dissociation attributed to the fast phase (%Fast) was calculated by fitting the dissociation traces to a biphasic model. The fit is indicated by the gray dotted lines. The overlaying data points in light gray are shown for 0 nM (small circles on top curve) and 10 nM (large squares on bottom curve) ISRIB only. Shown are traces from a representative experiment performed three times. Right: plot of the %Fast of the dissociation reactions to the left, as a function of ISRIB concentration. The plot was fitted to an [Agonist] versus response (Hill slope = 1) non-linear regression model (dotted line) yielding an EC50 of 1.8 (95% CI, 0.8–2.8) nM.
See also Figure S4.