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. 2021 Dec 7;12:7102. doi: 10.1038/s41467-021-27337-x

Fig. 2. Aromatic clusters of SFSV NSs are important for binding to eIF2B and suppressing the inhibitory effect of eIF2(αP).

Fig. 2

a MST analysis between eIF2B and SFSV NSs. Fluorescently labeled SFSV NSs (100 nM) was mixed with an equal volume of a 16-step serial dilution of 6.8 μM eIF2B and the microscale thermophoresis was measured. Plots of the wild-type SFSV NSs (maroon) and the alanine-substituted mutants on aromatic cluster 1 (SFSV NSs-c1-Ala-mut: Y5A, F7A, and F33A; yellow), aromatic cluster 2 (SFSV NSs-c2-Ala-mut: Y79A and F80A; blue), and both clusters (SFSV NSs-c1 + 2-Ala-mut: Y5A, F7A, F33A, Y79A, and F80A; cyan) are shown. Data are presented as mean values ± SDs at each concentration, and n = 3 independent experiments. b Two aromatic clusters of SFSV NSs at the interface with eIF2B. Aromatic cluster 1 (c1: Y5, F7, and F33) and aromatic cluster 2 (c2: Y79 and F80) grasp the α3 helix of the eIF2Bα subunit from both sides and bury the space between the helices of the eIF2Bα subunit. c–e Guanine nucleotide exchange assay. Non-phosphorylatable eIF2(αS51A) (final 150 nM) was loaded with BODIPY-GDP and fluorescent signals were read every 20 s. In the three panels, the gray lines are the measurements without eIF2B, and other measurements were started by the addition of eIF2B (final 40 nM). The red and green lines are the measurements without and with eIF2(αP) (final 1.5 μM), respectively. In the experiments shown in c, various concentrations (0–200 nM) of wild-type SFSV NSs were included in the reaction solutions containing fluorescent-eIF2(αS51A) and eIF2(αP). In the experiments shown in (d and e), ISRIB (d) or the mutants of SFSV NSs (c1-Ala-mut, c2-Ala-mut, and c1 + 2-Ala-mut) (e) were included into the reaction solutions at 200 nM. The same controls (No eIF2B, eIF2B, eIF2B + eIF2(αP) and eIF2B + eIF2(αP) + SFSV NSs at 200 nM) were used in (d and e). Data are presented as mean values ± SDs at each time point [n = 3 for eIF2B + eIF2(αP) + SFSV NSs at 100 nM in c, n = 4 for eIF2B + eIF2(αP) + SFSV NSs at 20, 40, 60 nM in (c), eIF2B + eIF2(αP) in (d and e), eIF2B + eIF2(αP) + SFSV NSs-c1-Ala-mut, eIF2B + eIF2(αP) + SFSV NSs-c1 + 2-Ala-mut in (e), and n = 5 for the rest of the experiments. n means the number of independent experiments]. Source data are provided as a Source Data file.