Structural basis for assembly and disassembly of the CRM1 nuclear export complex - PubMed (original) (raw)

Structural basis for assembly and disassembly of the CRM1 nuclear export complex

Xiuhua Dong et al. Nat Struct Mol Biol. 2009 May.

Abstract

CRM1 (or exportin 1, Xpo1) transports proteins out of the cell nucleus through the nuclear pore complex. In the cytoplasm, GTP hydrolysis and consequent dissociation of Ran from CRM1 releases low-affinity substrates, while additional factors facilitate release of high-affinity substrates. Here we provide a model for human CRM1 export complex assembly and disassembly through structural and biochemical analyses of CRM1 bound to the substrate snurportin 1 (SNUPN, also called snuportin 1).

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Figures

Figure 1

Figure 1

Comparison of unliganded and SNUPN-bound states of CRM1. (a) Ribbon diagram of the CRM1–SNUPN complex (PDB 3GB8). CRM1 is shown in pink and its C-extension in magenta, and several of the 20 HEAT repeats (H1–H20) are labeled. The sIBB and NBD domains of SNUPN are yellow and green, respectively. (b,c) Orthogonal views of the 22-Å-resolution density map calculated from coordinates of SNUPN-bound CRM1 (b) and the EM map of unliganded CRM1 (c). Ribbon diagram of SNUPN-bound CRM1 (blue-red color ramp from N to C terminus) is also shown in b, and a red triangle marks the LR-NES–binding site. N-terminal, central and C-terminal portions of CRM1 in the EM map are numbered in c.

Figure 2

Figure 2

Positive cooperativity of substrate and Ran binding to CRM1. (a) The CRM1–SNUPN binary intermediate can accommodate RanGTP. Repeats H2–H7 of SNUPN-bound CRM1 were superimposed with H2–H7 of the CSE1–Kap60– RanGTP complex (PDB 1WA5). CRM1 and SNUPN are colored as in Figure 1 and RanGTP is in blue. CSE1 and Kap60 are omitted for clarity. (b) The electrostatic surface potential of SNUPN-bound CRM1 is shown with RanGTP from the superimposed CSE1–Kap60–Ran complex. The basic patch of Ran (residues 129–142) is in purple, the H9 loop sequence is shown and a dashed line indicates the disordered portion of this loop. (c) Pulldown assays with immobilized GST-NES of HIV1-REV and CRM1 proteins in the presence or absence of RanGTP and/or leptomycin B (LMB) were visualized by SDS-PAGE and Coomassie blue staining.

Figure 3

Figure 3

Comparison of CRM1-, Kap-β1- and m3G-cap-nucleotide-bound SNUPN proteins. (a) The m3G-cap binding site in the CRM1–SNUPN complex. The m3G-cap nucleotide from the superimposed structure of nucleotide-bound NBD is shown as sticks (PDB 1XK5). CRM1 is shown as a pink ribbon and the sIBB and NBD of SNUPN as yellow and green ribbons, respectively. (b) Pulldown assays using immobilized GSTSNUPN and CRM1 were performed in the presence of RanGTP, m3G-cap nucleotide or Kap-β1. Bound proteins were separated by SDS-PAGE and visualized with Coomassie blue staining. (c) The Kap-β1–sIBB complex (PDB 2P8Q) is shown with the molecular surface of Kap-β1 in gray and the sIBB as a yellow ribbon. (d) Ribbon diagram of CRM1-bound SNUPN, showing that most of the sIBB (yellow) interacts with its NBD (green). m3G-cap nucleotide from the superimposed structure of nucleotide-bound NBD is shown as sticks. The structure is drawn with residues 40–52 of the sIBB in the same orientation as in c.

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