In addition to its HEAT repeats CRM
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, Droxinostat 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.
Discussion Efficient nuclear export requires CRM1 to recognize a wide diversity of cargos with high affinity in the nucleus while averting adverse binding events in the cytosol. Previous studies identified two structural elements of CRM1 as critical for maintaining the low-affinity cytosolic state: the HEAT-9 loop and the C-extension (Dong et al., 2009a, Fox et al., 2011, Koyama and Matsuura, 2010, Monecke et al., 2013, Saito and Matsuura, 2012). The inhibitory action of the HEAT-9 loop is well characterized: by interacting with the B helices behind the NES-binding groove, the loop stabilizes the groove in a constricted conformation refractory to NES binding (Fox et al., 2011, Koyama and Matsuura, 2010, Monecke et al., 2013, Saito and Matsuura, 2012). Inhibition is relieved in the nucleus by RanGTP, which detaches the loop from the groove-defining repeats, allowing the groove to open. Stabilization of the auto-inhibitory HEAT-9 loop conformation by the C-tail (at least partly) explains why deletion or mutation of the C-tail enhances NES-binding affinity (Fox et al., 2011, Monecke et al., 2013, Saito and Matsuura, 2012). Notably, deleting the C-tail has a weaker effect on NES-binding than deleting the entire C-extension (Fox et al., 2011), suggesting an additional inhibitory function of the C-extension besides tail-mediated effects. Regarding dynamic flexibility, NMA predicts that the WT conformation stabilized by the trans-ring C-extension is more rigid than that of CRM1ΔC, consistent with the latter’s decreased thermal stability. This agrees with molecular dynamics simulations reported for C. thermophilum CRM1, in which removal of the C-extension caused the solenoid to sample a larger range of conformations than the WT, indicating higher flexibility for the mutant (Monecke et al., 2013). The C-terminal arch stabilized by the C-extension includes the A helices that define the NES-binding groove and the B helices that stabilize the auto-inhibitory HEAT-9 loop conformation. The accordion-like motion of these helices described by the lowest-energy vibrational normal mode of CRM1ΔC (whereby the solenoid ring alternately opens and closes) provides an intuitive feeling for how loss of the C-extension could facilitate opening of the NES-binding groove (directly via A and indirectly via B helix motions), although the actual atomic trajectories are likely to be more complex. More generally, our findings are in line with previous studies showing that HEAT-repeat flexibility is an important aspect of importin and exportin function (Conti et al., 2006, Forwood et al., 2010, Kappel et al., 2010, Zachariae and Grubmüller, 2006, Zachariae and Grubmüller, 2008). In particular, the hypothesis that CRM1’s C-extension inhibits NES binding by rigidifying the HEAT-repeat solenoid is reminiscent of how the binding of RanGTP is proposed to lock importin β into a more rigid conformation that impairs the binding of FG-repeat containing nucleoporins to the outer surface of the solenoid (Bayliss et al., 2000, Lee et al., 2005).