class="head no_bottom_margin" id="sec1title">IntroductionThe plasticity of intrinsically disordered proteins (IDPs) is thought to be key to their highly diverse roles in the eukaryotic interactome and a variety of vital processes such as transcription, epigenetic regulation mechanisms, and transport through nuclear pore complexes (NPCs) (). The central channel of the NPC is filled with phenylalanine-glycine-rich proteins, called FG-nucleoporins (FG-Nups) that are intrinsically disordered (). FG-Nups build up an approximately 30-nm-thick permeability barrier through which large molecules (>40 kDa) can only be shuttled when bound to a nuclear transport receptor (NTR) with passage times as fast as 5 ms (). Due to the intrinsic dynamics of the FG-Nups, even state-of-the-art electron tomographic studies are not able to visualize them within the central NPC channel, despite their millimolar concentrations (). Consequently, the molecular structure of the permeability barrier and its general mode of action are widely debated (for a review see ).The key to understanding the observed nucleocytoplasmic transport phenomena resides in a description of the binding mode between FG-Nups and NTRs, for which a molecular analysis of the FG-Nup⋅NTR interaction is a prerequisite. Our current understanding of the molecular basis of FG-Nup⋅NTR interactions is in large part derived from X-ray crystallographic structures or molecular dynamics (MD) simulations of NTRs in the presence of short FG-peptides (up to ∼13 amino acids in length) (), as well as binding measurements with different NTRs or mutated NTR binding pockets (). Even for FG-Nups alone, only overall chain dimensions or long-range interactions within the Nups have so far been analyzed in solution (). Notably, even such fundamental binding characteristics as the equilibrium dissociation constant (Kd) between Nups and NTRs are still matter of discussion - estimates range from a few nM to several mM (). However, high Kd (low affinity, ∼mM) values are not easily compatible with high specificity of the transport process, while low Kd values (∼nM range) cannot easily explain high transport rates, since these might be expected to correlate with long residence times whereas NTRs must encounter many FG-Nups while crossing the thick barrier.Fast protein binding also typically requires proper orientation of the protein binding partners as well as conformational adaption of the IDP to bind to a folded protein. Those can occur prior to or during binding, as described by either of the two prevalent models for protein binding namely conformational selection and induced fit (). While such a conformational shift or fit can present the rate-limiting step of binding, fast binding is warranted in many biological processes. Several binding rate enhancing effects have been suggested or observed experimentally, such as maintenance of a degree of disorder (termed “fuzziness”; ) by conformational funneling (), a large capture radius of the flexible IDPs (), and the involvement of long-range electrostatic interactions to steer (attract) proteins together ().In this work, we characterize the conformational plasticity of Nups from human and yeast in the presence of structurally and functionally diverse NTRs. A focus was a PxFG-rich domain of the Nup153 (Nup153FGPxFG) as its size permitted a combination of nuclear magnetic resonance (NMR), single molecule Förster resonance energy transfer (smFRET), and molecular dynamics (MD) simulations to characterize local, residue specific, as well as long-range implications of Importinβ binding to Nup153FGPxFG conformation and dynamics. Additional Brownian dynamics (BD), fluorescence stopped-flow and single molecule transport experiments with functional NPCs in permeabilized cells, revealed the detailed kinetics of the complex formation between Nup and NTR. Using this molecular, integrative structural biology approach we propose a mechanism whereby Nups contribute low-affinity minimalistic binding motifs that act in concert to create a polyvalent complex. The global Nup structure and dynamics are largely unaffected by the interaction, thereby ensuring ultrafast binding and unbinding of individual motifs—a result that explains how nuclear transport can be fast yet specific, and that may have general implications for the mechanism of action of other IDPs that exhibit a multiplicity of binding motifs.
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