Thermodynamic characterization of the multivalent interactions underlying rapid and selective translocation through the nuclear pore complex.

Intrinsically disordered proteins (IDPs) play important roles in many biological systems. Given the vast conformational space that IDPs can explore, the thermodynamics of the interactions with their partners is closely linked to their biological functions. Intrinsically disordered regions of Phe-Gly nucleoporins (FG Nups) that contain multiple phenylalanine-glycine repeats are of particular interest, as their interactions with transport factors (TFs) underlie the paradoxically rapid yet also highly selective transport of macromolecules mediated by the nuclear pore complex. Here, we used NMR and isothermal titration calorimetry to thermodynamically characterize these multivalent interactions. These analyses revealed that a combination of low per-FG motif affinity and the enthalpy-entropy balance prevents high-avidity interaction between FG Nups and TFs, whereas the large number of FG motifs promotes frequent FG-TF contacts, resulting in enhanced selectivity. Our thermodynamic model underlines the importance of functional disorder of FG Nups. It helps explain the rapid and selective translocation of TFs through the nuclear pore complex and further expands our understanding of the mechanisms of "fuzzy" interactions involving IDPs.

Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations.

Protein-protein interactions are central to biological processes. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions between nucleocytoplasmic transport factors and intrinsically disordered FG repeat containing nuclear pore complex proteins as a model system, we examined the utility of three surface-based methods: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the apparent effect of mass transport limitations was demonstrated. Additional experiments with a loss-of-interaction FG repeat mutant variant demonstrated that the binding events that take place on surfaces can be unexpectedly complex, suggesting particular care must be exercised in interpretation of such data.