Transport factors in the karyopherin-β (also called importin-β) family mediate the movement of macromolecules in nuclear-cytoplasmic transport pathways. Karyopherin-β2 (transportin) binds a cognate import substrate and targets it to the nuclear pore complex. In the nucleus, Ran·GTP binds karyopherin-β2 and dissociates the substrate. Here we present the 3.0 A structure of the karyopherin-β2-Ran·GppNHp complex where GppNHp is a non- hydrolysable GTP analogue. Karyopherin-β2 contains eighteen HEAT repeats arranged into two continuous orthogonal arches. Ran is clamped in the amino- terminal arch and substrate-binding activity is mapped to the carboxy- terminal arch. A large loop in HEAT repeat 7 spans both arches. Interactions of the loop with Ran and the C-terminal arch implicate it in GTPase-mediated dissociation of the import-substrate. Ran GppNHp in the complex shows extensive structural rearrangement, compared to Ran·GDP, in regions contacting karyopherin-β2. This provides a structural basis for the specificity of the karyopherin-β family for the GTP-bound state of Ran, as well as a rationale for interactions of the karyopherin-Ran complex with the regulatory proteins ranGAP, ranGEF and ranBP1.
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