|dc.description.abstracteng||Transport into and out of the nucleus occurs through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope. NPCs are massive macromolecular complexes of around 125 kDa in vertebrates. Around 30 different nucleoporins (Nups) build the NPC, a third of which contain phenylalanine glycine (FG)-repeats (FG-Nups). The FG-repeats form the permeability barrier of the NPC, thereby restricting diffusion for most molecules larger than 20-40 kDa. In order for larger molecules to translocate through the NPCs, they have to be actively transported in complexes with transport receptors. Transport receptors were shown to interact with FG-repeats of nucleoporins, consequently overcoming the permeability barrier of the NPCs. The most prominent transport receptor for nuclear import is importin b, whereas CRM1 is the major nuclear export receptor. With the exception of some crystal structures of importin b with short, mostly chemically synthesized, FG-repeat containing peptides, structural information on how transport receptors interact with FG-Nups to mediate translocation through the NPC has not been available so far.
In this work, the interactions of CRM1 export complexes with Nup214 were analyzed by structural and biochemical methods. A complex containing CRM1, RanGTP, SPN1 and a Nup214(1916-2033) fragment was analyzed by cross-linking mass spectrometry and X-ray crystallography. The crystal structure revealed three FG-sites on the Nup214 fragment that bound to CRM1 by winding along the outer ring from HEAT-repeat 14, across the interacting region of the N- and C- termini, through to HEAT-repeat 4. Seven phenylalanine residues could be identified in Nup214 that bound to hydrophobic pockets on CRM1. Moreover, the residues between the bound phenylalanine residues seemed to be flexible or bound rather loosely to the CRM1 surface. Structure-derived Nup214 and CRM1 mutants were analyzed for their binding properties. Analysis of CRM1 mutants revealed that mutagenesis in several regions of CRM1 can affect its cooperative binding to RanGTP and NES. More than 90% of the CRM1 mutants designed to decrease binding to Nup214, also showed altered binding to RanGTP and an NES peptide. Two CRM1 mutants displayed decreased binding to Nup214 but remained unaltered with respect to RanGTP and NES binding, verifying that the hydrophobic pockets on CRM1 binding to Nup214 residues F1938 and F1982 in the crystal structure are indeed involved in the interaction. Correspondingly,
mutagenesis of the Nup214 phenylalanine residues bound in the structure clearly
reduced binding to CRM1. The obtained crystal structure of the Nup214 complex is the first structure of a karyopherin with long fragments of a FG-nucleoporin. The crystal structure combined with data from binding and transport assays suggests that binding sites of FG-nucleoporins on CRM1 partially, but not completely, overlap. Furthermore, Nup214 stabilizes CRM1 export complexes by acting as a molecular clamp.||de