![]() Other HEAT-motif-rich proteins (such as PPP2R1A and CAND1) can also pass though the NPC by themselves ( Yoshimura et al., 2014), indicating that the HEAT motif is generally suitable for NPC passage. These findings explain how karyopherins can overcome the hydrophobic NPC barrier. The affinity of karyopherins to FG-Nups is also known to be modulated by the structural changes induced by binding to their cargos ( Lott et al., 2010). Hydrophobic residues within the NPC induce conformational changes on karyopherins to expose more hydrophobic residues on the molecular surface, allowing the molecule to diffuse through the pore. Substitution of these residues with polar or charged residues reduces the affinity of karyopherins for FG-Nups. Karyopherins bind firmly to FG-Nups through hydrophobic pockets between adjacent A-helices ( Bayliss et al., 2000, 2002 Liu and Stewart, 2005 Otsuka et al., 2008). Previous structural analyses have revealed flexible structural changes of importin-β upon binding to its cargo ( Cingolani et al., 2000) or under different solvent conditions ( Yoshimura et al., 2014), and its conformational flexibility has been shown to be important for protein–protein interactions and nuclear translocation ( Lee et al., 2000). The structural flexibility of karyopherins is also important for their passage through the pore. A long array of HEAT motifs (HEAT repeat) confers the entire molecule with structural flexibility, which plays important roles in its interactions with cargo proteins and RanGTP ( Chook and Blobel, 2001 Conti et al., 2006 Forwood et al., 2010 Lee et al., 2000 Stewart, 2007). This characteristic helical composition makes the entire karyopherin molecule amphiphilic, with the hydrophobic sides facing each other toward the inside of the molecule and the hydrophilic sides facing outward to the solvent ( Fig. 1A). Karyopherins are composed of 19–21 tandem repeats of HEAT (Huntingtin, elongation factor 3, protein phosphatase 2A and PI3-kinase TOR1) motifs, each containing two amphiphilic α-helices (A-helix and B-helix) connected by a short linker region ( Chook and Blobel, 1999 Cingolani et al., 1999). The crystal structures of karyopherin-β family proteins exhibit significant similarities in their overall molecular shape, although their amino acid sequence similarities are very low (15–20% identity) ( O'Reilly et al., 2011 Xu et al., 2010). Karyopherin-β family proteins are designed to pass through the amphiphilic matrix of the NPC. In vivo, a strong hydrophobic crowding barrier has been shown to form in the NPC, which is especially enriched at both peripheries of the pore ( Konishi et al., 2017). This unique amino acid composition enables FG-Nups to form a hydrogel, where hydrophobic interactions between the phenylalanine residues crosslink the disordered polypeptides into a gel-like matrix ( Frey and Gorlich, 2006 Mohr et al., 2009). ![]() FG-Nups are similar to other intrinsically disordered proteins in that they are rich in polar residues, such as serine and threonine, but distinct from them by the frequent presence of hydrophobic residues (phenylalanine). The central channel of the NPC comprises a number of unstructured (disordered) polypeptides carrying phenylalanine-glycine (FG) motifs (FG-Nups). The entire complex is composed of more than 30 different Nups, each of which exists as multiple copies. Many approaches using biochemistry, structural biology and bioinformatics have revealed the structure of the NPC and the properties of its components, called nucleoporins (Nups) ( Cronshaw et al., 2002 Rout et al., 2000). These findings reveal the essential roles of prolines in ensuring the structural flexibility and functional integrity of karyopherins. However, in vitro and in silico analyses revealed that the mutant lost structural flexibility and could not undergo rapid conformational changes when transferring from a hydrophilic to hydrophobic environment or vice versa. These mutations did not alter the overall folding of the helical repeat or affect its interaction with cargo or the regulatory factor Ran. ![]() When these prolines were substituted with alanine, nuclear transport activity was reduced drastically in vivo and in vitro, and caused a severe defect in mitotic progression. Here, we focused on the characteristic enrichment of prolines in the middle of the outer α-helices of importin-β. The karyopherin family of nuclear transport receptors is composed of a long array of amphiphilic α-helices and undergoes flexible conformational changes to pass through the hydrophobic crowding barrier of the nuclear pore.
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