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

Ryo Hayama,Samuel Sparks,Lee M Hecht,Kaushik Dutta,Jerome M Karp,Christina M Cabana,Michael P Rout,David Cowburn

doi:10.1074/jbc.ac117.001649

Abstract

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.

Highlights

Disordered proteins (IDPs) play important roles in many biological systems
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 highly selective transport of macromolecules mediated by the nuclear pore complex
The binding of multiple FG motifs to a TF containing multiple interaction sites could lead to high avidity complexation and long residence times, and such interactions would be incompatible with the rapid transport rates observed in vivo dered region; SLiM, short linear motif; FG Nup, Phe–Gly nucleoporin; HSQC, heteronuclear single quantum coherence; DLS, dynamic light scattering

Summary

Results

We characterized the interaction between FG Nups and NTF2 with a series of constructs containing a variable number of FSFG motifs by sequentially replacing FSFG motifs with SSSG motifs (Fig. 1B). This excludes an interaction mechanism where each FSFG motif binds independently to a different molecule of NTF2, where no change in the measured affinity would be expected. This trend again reflects the additive nature of the system [30] (i.e. the frequency of qualitatively similar contacts increases with valency) We conclude that this modest avidity is maintained because the effect of increased local concentration of FSFG motifs around NTF2 is countered by the entropic costs of restricting the conformational freedom of the chain, preventing stable multibound states (Fig. 4D)

Discussion

Experimental procedures

Protein purification

Nuclear magnetic resonance

Isothermal titration calorimetry