Decoding Protein Plasticity from Single Molecules to Large Complexes

Abstract

Intrinsically disordered and phenylalanine-glycine rich nucleoporins (FG-Nups) form a selective permeability barrier inside the nuclear pore complex (NPC): Large molecules can only cross the central channel of the NPC when piggybacked by nuclear transport receptors (NTRs) that specifically interact with FG-Nups. These FG-Nups, however, display complex and non-random amino acid architecture and possess repeatedly occurring FG-motifs flanked by distinct amino acid stretches. How such heterogeneous sequence composition relates to function and how homotypic interactions between such disordered stretches, and transient heterotypic interactions with folded transport receptors could give rise to a transport mechanism is still unclear. To address this challenge we developed a combined fluorescence correlation and time resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to NTRs. The detection of segmental backbone mobility of Nup153 within the unperturbed complex provides a readout of local, region specific, binding properties that are usually masked in measurements of the whole IDP. Binding affinity of functionally and structurally diverse NTRs to distinct regions of Nup153 differed by orders of magnitudes - a result with implications for the diversity of transport routes in nucleocytoplasmic transport. Furthermore, synergistic molecular dynamics simulations permitted visualization of previously unknown steps and binding modes during Nup•NTR interactions at atomic resolution. These results have molecular implications for the diversity of transport routes within nucleocytoplasmic transport and on how nuclear transport can pursue specifically and very fast inside the nuclear pore complex.