title: Promiscuous binding of Karyopherinβ1 modulates FG nucleoporin barrier function and expedites NTF2 transport kinetics.
abstract: The transport channel of nuclear pore complexes (NPCs) contains a high density of intrinsically disordered proteins that are rich in phenylalanine-glycine (FG)-repeat motifs (FG Nups). The FG Nups interact promiscuously with various nuclear transport receptors (NTRs), such as karyopherins (Kaps), that mediate the trafficking of nucleocytoplasmic cargoes while also generating a selectively permeable barrier against other macromolecules. Although the binding of NTRs to FG Nups increases molecular crowding in the NPC transport channel, it is unclear how this impacts FG Nup barrier function or the movement of other molecules, such as the Ran importer NTF2. Here, we use surface plasmon resonance to evaluate FG Nup conformation, binding equilibria, and interaction kinetics associated with the multivalent binding of NTF2 and karyopherinβ1 (Kapβ1) to Nsp1p molecular brushes. NTF2 and Kapβ1 show different long- and short-lived binding characteristics that emerge from varying degrees of molecular retention and FG repeat binding avidity within the Nsp1p brush. Physiological concentrations of NTF2 produce a collapse of Nsp1p brushes, whereas Kapβ1 binding generates brush extension. However, the presence of prebound Kapβ1 inhibits Nsp1p brush collapse during NTF2 binding, which is dominated by weak, short-lived interactions that derive from steric hindrance and diminished avidity with Nsp1p. This suggests that binding promiscuity confers kinetic advantages to NTF2 by expediting its facilitated diffusion and reinforces the proposal that Kapβ1 contributes to the integral barrier function of the NPC.
authors.affiliation: Biozentrum and the Swiss Nanoscience Institute, University of Basel, Basel, Switzerland.
authors.forename: Raphael S
authors.initials: RS
authors.lastname: Wagner
authors.affiliation: Biozentrum and the Swiss Nanoscience Institute, University of Basel, Basel, Switzerland.
authors.forename: Larisa E
authors.initials: LE
authors.lastname: Kapinos
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Marshall
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Murray
authors.initials: M
authors.lastname: Stewart
authors.affiliation: Biozentrum and the Swiss Nanoscience Institute, University of Basel, Basel, Switzerland. Electronic address: roderick.lim@unibas.ch.
authors.forename: Roderick Y H
authors.initials: RYH
authors.lastname: Lim
mesh_terms: D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*
chemical_list: C424166:KPNB1 protein, human; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C503612:Nutf2 protein, rat; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins