NMR chemical shift mapping reveals the binding site for MNV VPg on the eIF4GI HEAT-1 domain.

(A) Superposition of the ¹H¹⁵N TROSY HSQC spectra of ¹⁵N-labelled eIF4GI HEAT-1 (748–993) obtained in the absence (black) and presence (green) of 0.35 molar equivalents of unlabelled MNV VPg(104–124) peptide. (¹H¹⁵N TROSY NMR spectra for all of the points in the titration are given in S3 Fig). The residues for which the intensity of amide ¹H¹⁵N signals exhibit the greatest reductions as the MNV VPg(104–124) peptide concentration is increased are indicated by their residue number. The assignments were taken from the Bio Magnetic Resonance Database (BMRB id 18738). (B) Plot of the ratio of the absolute values of non-overlapping peak volumes for assigned residues obtained at 0 and 0.35 molar equivalents of the MNV VPg(104–124) peptide. High values of the ratios indicate the residues in eIF4GI HEAT-1 most affected by MNV VPg binding. The red dashed line indicates the expected volume ratio (R_v) for unperturbed residues: 0.76, based on the relative number of scans performed in each HSQC experiment. Points above the green dashed line indicate residues for which the peak volume ratio was at least six times higher than the baseline for unperturbed amides. (C) Model of the complex of eIF4GI HEAT-1 (grey, surface representation) and eIF4A (cyan, cartoon representation) indicating the predicted location of the VPg binding site. Surface residues in eIF4GI HEAT-1 that exhibit the greatest changes in R_v in the presence of the MNV VPg(104–124) peptide are coloured yellow. The model of eIF4GI HEAT-1 was generated using SWISS-MODEL [46]; the complex was created by superposing this model on the eIF4G HEAT-1 component of the yeast eIF4G-eIF4A co-crystal structure [47]. (D) SDS PAGE analysis of pull-down assays to test the effect of mutations in the putative VPg-binding site on eIF4G HEAT-1 on binding to the viral protein. His-tagged MNV VPg(1–124) was used as bait; eIF4GI HEAT-1 wild-type or mutant proteins were used as prey, and the pull-down buffer contained 150 mM NaCl (top panel) or 300 mM NaCl (bottom panel). Lanes 1–8 (blue labels): input protein mixtures; lanes 9–16 (red labels): eluted proteins. (E) Graphical representation of the results of the cobalt affinity pull-down assays performed in the presence of 150 or 300 mM NaCl, plotted as the ratio of the optical densities of the eIF4GI HEAT-1 band to that of the His-MNV VPg band in eluted fractions (lanes 9–16 in panel D). Band densities were quantified using ImageJ (http://imagej.nih.gov/ij/) and the ratios were normalised to the wild-type control.