Fig. 6: Unliganded Msn5 and the mechanisms for loading/unloading Pho4.
From: Phosphate-dependent nuclear export via a non-classical NES class recognized by exportin Msn5
a Unliganded Msn5 (gray, left) and Msn5-RanGTP-pPho41-200 (gray-teal-wheat, right). Structural differences at h15loop, h17 and C-helix-tail are highlighted by yellow, purple and green dashed lines, respectively. The h4-h5loop that contacts h19-h20 in unliganded Msn5 but not in ligand-bound Msn5, is highlighted with black dashed lines. b FP titration of Msn5 WT or mutant ΔC-helix-tail to mNeonG-RanGTP ± excess pPho41-200. Data points are mean and s.d. of triplicate measurements, fitted with 1-site fitting (solid line) and fitting residuals are plotted below. c Bound proteins (after extensive washing) in an in vitro pull-down assay of immobilized unphosphorylated (-P) or phosphorylated GST-Pho41-200 ( + P) incubated with Msn5 (WT or ΔC-helix-tail) ± RanGTP, visualized by Coomassie stained SDS-PAGE. Gel image from the single experiment is shown. d Msn5 (gray; h17 residues 960-979 in dark gray)-RanGTP-pPho41-200 and CRM1-RanGTP-RanBP1 (3M1I [https://doi.org/10.2210/pdb3M1I/pdb]) structures aligned at RanGTP. CRM1 and pPho4 are not displayed for clearer visualization of the docked RanGTP-RanBP1 (aquamarine and pink, respectively). e As in c, pull-down assay of GST-pPho41-200 incubated with Msn5 WT or Δh17loop (residues 962-980 replaced with GGSGGS) ± RanGTP FL ± RanBP1 as labeled. Gel image from the single experiment is shown. f Cartoon model of cargo loading and unloading. Source data for b, c and e are provided as a Source Data file.
