Crystal structures of various CRM complexes have provided

Crystal structures of various CRM1 complexes have provided insight into molecular details of the interactions between CRM1 and its interaction partners during the transport cycle. CRM1 cooperatively binds RanGTP and cargo in the nucleus (Paraskeva et al., 1999). In this ternary complex, RanGTP is localized within the ring of CRM1 and bound by N- and C-terminal HEAT repeats as well as the acidic loop (AL). The AL is inserted between the helices of HEAT repeat 9 (H9) and affixes the GTPase to the terminal HEAT repeats like a seatbelt (Monecke et al., 2009). Remarkably, the cargo binds on the opposite side of CRM1 without direct contacts to RanGTP. It predominantly interacts with acidic patches on the outer surface of CRM1 and a groove formed between the α helices of H11 and H12 (Dong et al., 2009a, Dong et al., 2009b, Monecke et al., 2009). A common motif required for binding of cargo within this groove is the leucine-rich nuclear export signal (NES) consisting of a short peptide stretch of 10–15 residues (Güttler et al., 2010). The CRM1-RanGTP-cargo complex traverses the NPC and enters the cytosol, where it miniprep dissociates upon binding of disassembly factors such as RanBP1, which function as cargo release factors and increase the hydrolysis rate of Ran-bound GTP when binding to RanGAP (Askjaer et al., 1999, Koyama and Matsuura, 2010, Maurer et al., 2001, Paraskeva et al., 1999). Free CRM1 shuttles back to the nucleus for another round of export. The reported crystal structures reveal snapshots of various states during the transport process and show the interaction surfaces of CRM1 with cargo and/or Ran. Due to the growing medical interest in CRM1 and its role in cancer (Turner et al., 2012), it is important to understand the dynamics of human CRM1 with a focus on the NES-binding cleft where many therapeutics bind. Purely static structure characterization alone is insufficient for a complete description of structural changes during the transport cycles. Recent findings from MD simulations on the free—extended—form of CRM1 from the lower eukaryote C. thermophilum, have shown that the α helix of H21, but not the AL, contribute significantly to the ratio between the extended and the compact form of CRM1 (Monecke et al., 2013). In the ternary complex of CRM1-RanGTP-SPN1, the altered arrangement of the AL, bridging the central opening and linking two distant regions of CRM1, suggests a structural role for determining both the overall conformation of CRM1 and that of the NES-binding cleft. Moreover, the role of RanGTP in restricting the conformational flexibility of CRM1, especially regarding the NES-binding cleft, and the opening mechanism of the toroidal form of CRM1 toward the extended conformation are still open questions. Here, small-angle X-ray scattering (SAXS), electron microscopy (EM), and molecular dynamics (MD) simulations were combined with the available information from crystal structures to elucidate the structural transitions and forces required for the cooperative binding and release of RanGTP and/or the cargo Snurportin1 (SPN1) to mammalian CRM1. We find that mammalian CRM1 in the free form reveals a high degree of conformational flexibility. Binding of RanGTP decreases this flexibility and shifts the conformation toward a more rigid, compact form of CRM1. Our results also show that the AL has a strong influence over the state of the NES-binding cleft. We conclude that RanGTP binding in the presence of the AL ensures that the NES-binding cleft for export cargo remains in an open conformation prone for NES binding, and thus enhances the affinity for cargo.