Plasticity of an Ultrafast Interaction between Nucleoporins and Nuclear Transport Receptors

Abstract

SummaryThe mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

“…3C). The FG motif residues, which contribute most strongly to the interaction with NTF2, displayed elevated R 2 values indicative of slower motion of these interacting residues, consistent with previous studies (29,30). We expect contributions from chemical exchange (R ex ) to be limited as previously noted (29).…”

“…Both transport factors were predicted to have multiple interaction sites for FG repeats; these results agree with NMR studies (29,34). More recently, multiple short simulations of FG repeats were performed in the presence of TFs (30). In these simulations, FG repeats bound readily to TFs, but formed a more compact state than expected based on smallangle X-ray scattering (SAXS) data obtained in the same study (35).…”

“…Our recent NMR measurements have demonstrated that the FG repeats of FSFG 6 undergo rapid exchange at their interaction sites on Kap95 (29); this rapid exchange was also reported for a different sequence of FG repeats from hNup153 during the interaction with several TFs of different structural architectures, including hNTF2, importin-β, and hTRN1 (30). These recent NMR measurements also showed that FG repeats remain highly disordered while transiently interacting with TFs (29,30). However, the thermodynamics and kinetics of this rapid exchange are not yet understood.…”

“…Several mesoscale models have been proposed to explain the transport mechanism (21), though none describes FG-TF interactions at the atomic level of detail. Our previous experimental studies (29), as well as independent studies from other groups (30), indicate that TFs associate and dissociate from FG motifs at an exceptionally fast rate. This fast exchange may reconcile the apparent discrepancy between transport that is rapid on the one hand, and selective on the other (Fig.…”

“…Such high-speed FG-TF interactions have been recently observed by stopped-flow measurements (30). However, experimental methods are typically unable to provide details of local interaction site dynamics at atomic resolution and at multiple timescales, spanning a range from individual molecular interactions to the entire transport event.…”