American Society of Hematology

Figure 2

$Figure 2. Comparison of the inhibitor and NES-bound grooves. (A) Sequence alignment of NES-binding grooves (HEAT repeats H11 and H12) of S cerevisiae XPO1 and human XPO1. Identical residues are shaded in gray, residues that contact KPT-185 are marked with black asterisks, and residues that contact the PKINES (3NBY) are marked with red asterisks. (B) Superposition of the KPT-185 (pink) and PKINES-bound (green) grooves. KPT-185 (orange) and Cys539 of ScXPO1* (pink) are shown as sticks. (C) Same view as in (B), but rotated 90° about the vertical axis and helices H12A of both grooves were removed to obtain a clear side view of the ligands in the groove. The PKINES and its hydrophobic sidechains are colored bright green. (D-E) Surface representations of the KPT-185 (D) and PKINES-bound XPO1 grooves (E). Distances across the openings of the grooves are shown in red. The 13-residue long PKINES peptide is substantially larger than KPT-185 and occupies the entire groove, burying 1117 Å2 whereas KPT-185 buries only 420 Å2 of the XPO1 groove. When the PKINES and KPT-185–bound grooves are superimposed, it is obvious that hydrophobic residues 2, 3, and 4 of the peptide overlap with the inhibitor. Two overlaps with methoxy group, 3 with the triazole, and 4 overlaps with the terminal oxadiazole group of KPT-185.(F) KPT-185 inhibits XPO1-cargo interactions. Approximately 15 μg of GST-NESs were immobilized on glutathione sepharose and then incubated with 10μM XPO1 proteins that were preincubated with either buffer or inhibitors (20μM LMB or 200μM KPT-185) and molar excess of RanGTP. After extensive washing, a fraction of the bound proteins was visualized by SDS-PAGE and Coomasie blue staining. (G) HeLa cells expressing Rev-BFP and/or wild-type XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP localizes in the nucleoli of the cells, whereas XPO1-YFP is mainly found at the nuclear rim. In cells coexpressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and colocalizes with Rev-BFP. Two hours after addition of SINEs the colocalization of XPO1-YFP with Rev-BFP in the nucleoli was analyzed. Both compounds disrupt the wild-type XPO1-YFP colocalization with Rev-BFP, although they had no effect when a mutant XPO1-YFP (C528S) was used as shown in panel H.$

Comparison of the inhibitor and NES-bound grooves. (A) Sequence alignment of NES-binding grooves (HEAT repeats H11 and H12) of S cerevisiae XPO1 and human XPO1. Identical residues are shaded in gray, residues that contact KPT-185 are marked with black asterisks, and residues that contact the ^PKINES (3NBY) are marked with red asterisks. (B) Superposition of the KPT-185 (pink) and ^PKINES-bound (green) grooves. KPT-185 (orange) and Cys539 of ^ScXPO1* (pink) are shown as sticks. (C) Same view as in (B), but rotated 90° about the vertical axis and helices H12A of both grooves were removed to obtain a clear side view of the ligands in the groove. The ^PKINES and its hydrophobic sidechains are colored bright green. (D-E) Surface representations of the KPT-185 (D) and ^PKINES-bound XPO1 grooves (E). Distances across the openings of the grooves are shown in red. The 13-residue long ^PKINES peptide is substantially larger than KPT-185 and occupies the entire groove, burying 1117 Å² whereas KPT-185 buries only 420 Å² of the XPO1 groove. When the ^PKINES and KPT-185–bound grooves are superimposed, it is obvious that hydrophobic residues 2, 3, and 4 of the peptide overlap with the inhibitor. Two overlaps with methoxy group, 3 with the triazole, and 4 overlaps with the terminal oxadiazole group of KPT-185.(F) KPT-185 inhibits XPO1-cargo interactions. Approximately 15 μg of GST-NESs were immobilized on glutathione sepharose and then incubated with 10μM XPO1 proteins that were preincubated with either buffer or inhibitors (20μM LMB or 200μM KPT-185) and molar excess of RanGTP. After extensive washing, a fraction of the bound proteins was visualized by SDS-PAGE and Coomasie blue staining. (G) HeLa cells expressing Rev-BFP and/or wild-type XPO1-YFP were analyzed by confocal fluorescence microscopy. Rev-BFP localizes in the nucleoli of the cells, whereas XPO1-YFP is mainly found at the nuclear rim. In cells coexpressing both Rev-BFP and XPO1-YFP, XPO1 is redistributed to the Rev-containing nucleoli and colocalizes with Rev-BFP. Two hours after addition of SINEs the colocalization of XPO1-YFP with Rev-BFP in the nucleoli was analyzed. Both compounds disrupt the wild-type XPO1-YFP colocalization with Rev-BFP, although they had no effect when a mutant XPO1-YFP (C528S) was used as shown in panel H.

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