Uncoupling Kapβ2 substrate dissociation and ran binding

Karyopherinβ2 (Kapβ2) imports a variety of mRNA binding proteins into the nucleus. Release of import substrates in the nucleus involves formation of a high-affinity Kapβ2-RanGTP complex and concomitant dissociation of import substrates. The crystal structure of the Kapβ2-RanGppNHp complex shows that Ran binds in the Kapβ2 N-terminal arch and substrate most likely binds its C-terminal arch. The structure suggested a mechanism for Ran-mediated substrate dissociation where a long internal acidic loop in Kapβ2 transmits structural information between the GTPase and substrate sites, leading to displacement of substrate by the loop when Ran is bound. To study the molecular mechanism of substrate dissociation, we have cleaved the acidic loop of Kapβ2 proteolytically (cl-Kapβ2) and also constructed a mutant of Kapβ2 with a truncated loop (TL-Kapβ2). Both modified Kapβ2s are unable to undergo Ran-mediated substrate dissociation. We have also mapped the boundaries of the Kapβ2 binding site of substrate mRNA binding protein A1 using a widely applicable method employing NMR spectroscopy. This has allowed design of reagents to quantitate the affinities of the Kapβ2 proteins for Ran and substrate. cl-Kapβ2, TL-Kapβ2, and native Kapβ2 have comparable affinities for both RanGppNHp and import substrates, indicating that perturbation of the loop has not altered the strength of binary Kapβ2-Ran or Kapβ2-substrate interactions. The TL-Kapβ2 mutant also binds RanGppNHp and substrate simultaneously to form a ternary complex, indicating that in addition to the loss of coupling between Ran binding and substrate dissociation, the two ligand sites on Kapβ2 are spatially distinct. The uncoupling of Ran binding and substrate dissociation in the TL-Kapβ2 mutant is further evident in significant loss of Ran-mediated nuclear uptake of fluorescent substrate in digitonin-permeabilized HeLa cells. These results support our previously proposed GTPase-mediated Kapβ2-substrate dissociation mechanism where the acidic loop of Kapβ2 physically couples distinct Ran and substrate binding sites.