Molecular mechanism of CRM1-mediated Nuclear Export

Orateur : Cyril DIAN (IMAXIO Biotech - Lyon - France)

The exportin CRM1 associates with the small GTPase Ran to mediate the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES), including many diverse cellular and viral cargos implicated in cancer and infectious disease. Export requires the cooperative assembly of a CRM1/RanGTP/NES complex in the nucleus and its dissociation in the cytosol. Crm-1 mediated nuclear export complex is also regulated by Ran Binding Proteins (RanBPs) that promotes either assembly or disassembly of the complex. Yet how cooperativity and cargo selectivity are achieved is poorly understood. To address this, we investigated human CRM1 in the unbound, cytosolic state and its cofactor RanBP3. We obtained the crystal structure and small-angle scattering analysis of a CRM1 mutant with enhanced NES-binding activity due to deletion of the C-extension. Structural and biochemical analysis confirmed that unbound CRM1 possesses a constricted (low-affinity) NES-binding groove stabilized by an auto-inhibitory loop involved in Ran recognition. We show that loss of the C-extension induces CRM1’s ring-shaped HEAT repeats to adopt a more extended spiral shape. Structure-based point mutations designed to destabilize the C-extension shift CRM1 to a more extended conformation, reduce thermal stability and enhance NES binding activity. Taken together these findings provide mechanistic insights into how the auto-inhibitory loop and the C-extension inhibit CRM1 from binding NES-bearing cargos in the cytosol. In addition, we solved the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. Crm1-mediated export is distinct from and of higher complexity than that observed for other exportins, whereby coordinated dynamic and conformational changes allow CRM1 to export a wide diversity of cargos while preserving the sharp RanGTP dependence required for efficient transport.

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