Investigation into the dynamic mechanism of nuclear transport through the nuclear pore complex

Abstract

Nuclear pore complexes (NPCs) are the sole mediators of nucleocytoplasmic exchange controlling the flow of molecules into and out of the nucleus. The selective filter properties of NPCs enable translocation of molecules known as transport factors (TFs) and their cognate cargo, while simultaneously preventing the passage of non-specific macromolecules. Central to this selectivity barrier is a group of largely intrinsically disordered nucleoporins (Nups) that contain multiple phenylalanine-glycine repeats, termed FG Nups. Somehow, the interactions between FG Nups and TFs enable TFs to translocate rapidly yet selectively through the NPC in what is referred to as the "transport paradox". To explain this paradox, multiple complementary approaches were used to characterize the molecular behaviors of FG Nups and their interaction with TFs. Results demonstrated that FG Nups are fully disordered, random coil polymers that remain disordered while engaged to TFs. FG Nups interact with TFs using predominantly their FG motifs and minimally their intervening spacer residues. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) studies demonstrated that the overall enthalpy of the interaction increases as multivalency increases the frequency of individually weak FG-TF contacts. However, tight binding is limited by an entropy penalty that disfavors simultaneous engagements of FG motifs from the same FG Nup. Small angle neutron scattering (SANS) additionally showed that the entropy loss is partly due to the local rigidity of an FG motif in the interacting state. Conformational ensembles derived from SANS data also indicate that an increase in the overall size of FG Nups is associated with TF interaction. Lastly, all-atom molecular dynamics (MD) simulation showed that spacers between the FG motifs behave as "entropic springs", disfavoring any static binding of the FG repeats. The dynamics of FG Nups enables the FG motifs to slide along the hydrophobic patches of TFs enabling FG motifs to be easily displaced by other competing FG motifs. This explanation provides a simple mechanism for the rapid exchange of TF-FG motif contacts during transport. Results from this thesis illuminated fundamental aspects of the functioning mechanisms underlying NPC transport at high structural resolution, something lacking in current models of nuclear transport.