Fig. 4.

(A) Restraint-driven model of the VPg (green) and eIF4E (blue) complex. Red highlights W56 from eIF4E involved in the association with VPg (SI Appendix, Fig. S8C). (B) Close-up view of the complex highlighting the positive patch on eIF4E and its interaction with negative residues on VPg (rotation of 120° compared with A). This region was confirmed to bind eIF4E by mutation (SI Appendix, Fig. S8 B and E). (C) Overlay of the HSQC spectra of ¹⁵N-labeled eIF4E (50 μM) in the presence of 3-fold excess of unlabeled VPg (blue) and after addition of 20-fold molar excess of m⁷GDP relative to eIF4E (orange), demonstrating that VPg and the cap compete for overlapping binding surfaces on eIF4E. (D) eIF4E specifically binds to VPg with submicromolar affinity (∼0.3 μM). Normalized change in fluorescence at emission of 484 nm (Δ[F]) as a function of eIF4E concentration for 0.5 μM VPgΔ37. Measurements were carried out 3 independent times.