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    • Atomic structures of mammalian and fungal CRM alone and in

    2020-07-30

    Atomic structures of mammalian and fungal CRM1, alone and in complex with different binding partners, have revealed that CRM1 undergoes multiple conformational changes as it shuttles between the nucleus, where cooperative assembly of the CRM1/Ran/cargo complex occurs, and the cytosol, where the complex disassembles (Dong et al., 2009b, Güttler et al., 2010, Koyama and Matsuura, 2010, Monecke et al., 2009, Monecke et al., 2013, Saito and Matsuura, 2012). Human CRM1 consists of 1,071 residues organized into 20 (or 21 depending on numbering convention; Dong et al., 2009b, Monecke et al., 2009) tandem HEAT repeats, each forming a hairpin of helices denoted A and B. The repeats define a ring-shaped solenoid, whose outer and inner surfaces comprise the A and B helices, respectively. Ran binds inside the ring, engaging the B helices of N- and C-terminal repeats and an acidic loop within HEAT-repeat 9. The NES binds to CRM1’s outer surface, occupying a groove formed by the A helices of repeats 11 and 12 (Dong et al., 2009b, Güttler et al., 2010, Monecke et al., 2009). The helical NES motifs of Snurportin1 (Spn1) and protein kinase A inhibitor (PKI) and the more extended NES motif of HIV-1 Rev dock into a common set of five TMPyP4 tosylate pockets within this groove (Güttler et al., 2010). The disassembly factor RanBP1 promotes cargo release from the CRM1/Ran/NES complex by inducing the HEAT-9 loop to switch from a conformation interacting with Ran to a conformation interacting with the B helices behind the NES-binding groove, causing the latter to constrict and extrude the bound NES (Koyama and Matsuura, 2010). In the absence of Ran, the HEAT-9 loop plays an auto-inhibitory role by maintaining the NES-binding groove in a constricted conformation incompatible with NES binding (Fox et al., 2011, Monecke et al., 2013, Saito and Matsuura, 2012). In addition to its HEAT repeats, CRM1 also possesses a C-terminal extension (C-extension, residues 1032–1071), composed of a helix (C-helix) followed by a short stretch of residues (C-tail). In the absence of Ran, the C-extension adopts a conspicuous conformation that bridges across the ring-shaped molecule to interact with central HEAT repeats and the HEAT-9 loop (Dong et al., 2009b, Monecke et al., 2013, Saito and Matsuura, 2012), whereas in the presence of Ran the C-extension is located on CRM1’s outer surface (Güttler et al., 2010, Koyama and Matsuura, 2010, Monecke et al., 2009). Deletion of the C-extension greatly enhances NES-binding affinity (Dong et al., 2009a, Fox et al., 2011), indicating an auto-inhibitory role for this element. Inhibition (at least partly) reflects stabilization of the auto-inhibitory conformation of the HEAT-9 loop by the C-tail, as seen in unliganded fungal CRM1 structures (Monecke et al., 2013, Saito and Matsuura, 2012). Interestingly, deletion of the entire C-extension enhances NES-binding to a greater extent than deletion of the C-tail (Fox et al., 2011), suggesting an additional inhibitory effect of the C-helix besides that mediated by the C-tail.