FIGURE 5.

NMR-determined chemical shift perturbations on schistosome eIF4E binding to m^2,2,7GpppG compared with m⁷GpppG. A, superposition of the ¹H-¹⁵N HSQC spectrum of the schistosome eIF4E bound to m⁷GpppG (red) and bound to m^2,2,7GpppG (blue). B, specific chemical shift perturbations on binding m^2,2,7GpppG compared with m⁷GpppG. The reported chemical shift Δδ represents the weighted chemical shift by applying the Pythagorean theorem: Δδ{¹H,¹⁵N} = {Δδ(¹H)² + 0.2 × Δδ(¹⁵N)²}^½ where Δδ(¹H) and Δδ(¹⁵N) are the chemical shift difference of the amide proton and nitrogen, respectively (28, 69). Amino acid residues with large chemical shifts are labeled. C and D, residues with major chemical shift perturbation on binding m^2,2,7G compared with the m⁷G cap mapped onto the crystal structure of the m⁷GpppG·eIF4E complex. The backbone residues with major chemical shifts are colored red. Residues with major chemical shifts in the vicinity of the second base of the cap are shown in C, and those with major chemical shifts in the vicinity of the third phosphate of the cap are shown in D. E, Clustal sequence alignment of several eIF4E illustrating conserved residues, residues undergoing CSP, and mouse pepsin cleavage sites on cap binding. The black boxes denote residues with major chemical shift perturbation in our NMR experiments as described above. The vertical black arrows denote the specific pepsin cleavage sites based on the mammalian eIF4E (21). Starred residues are those involved in cap binding with the two key Trp residues in red. The red box indicates two key residues for C. elegans eIF4E reported to be important for m^2,2,7G cap binding specificity (24). Note that C. elegans eIF4E-5 and Ascaris eIF4E-3 binds both types of caps, whereas mouse eIF4E3 and C. elegans eIF4E-3 are specific for m⁷G cap.