Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC.DOI: http://dx.doi.org/10.7554/eLife.10027.001

Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport.nuclear pore | molecular dynamics | nucleoporins | transport factors | NMR T he nuclear pore complex (NPC) regulates macromolecular transport between the nucleus and the cytoplasm in all eukaryotic cells (1, 2). The NPC allows for passive diffusion of small molecules including sugars, ions, and water, while simultaneously imposing size-dependent exclusion of macromolecules without nuclear transport factor (TF) binding sites, generating unique nuclear and cytoplasmic compartments (3, 4). Larger macromolecules can bypass the selectivity barrier of the NPC by interacting with TFs that shuttle cargo through the central channel of the pore. In pathological conditions, including many cancers and viral infections, the NPC is hijacked and used to transport undesired particles, or it is modified to attenuate the cellular responses to disease onset (5, 6).TFs traverse the NPC by selective and reversible association with disordered phenylalanine-glycine (FG) repeat domains of the FG nucleoporin proteins (FG Nups), which line the surface of the NPC (7-14). Each FG repeat domain consists of 5-50 FG repeats. In turn, each FG repeat consists of a single FG motif of various sequence flavors, such as the FSFG and GLFG motifs, and 10-30 spacer residues that separate consecutive FG motifs (15, 16). The spacer residues are strongly hydrophilic (17) and rich ...

The pathologic self-assembly of proteins is associated with typically late-onset disorders such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown, but there is increasing evidence supporting the role of transient α-helices in the early events. Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide that self-assembles into aggregates that are toxic to the insulin-producing β cells. To elucidate early events in the self-assembly of IAPP, we used limited proteolysis to identify an exposed and flexible region in IAPP monomer. This region includes position 20 where a serine-to-glycine substitution (S20G) is associated with enhanced formation of amyloid fibrils and early onset type 2 diabetes. To perform detailed biophysical studies of the exposed and flexible region, we synthesized three peptides including IAPP(11-25)WT (wild type), IAPP(11-25)S20G, and IAPP(11-25)S20P. Solution-state NMR shows that all three peptides transiently populate the α-helical conformational space, but the S20P peptide, which does not self-assemble, transiently samples a broken helix. Under similar sample conditions, the WT and S20G peptides populate the α-helical intermediate state and β-sheet end state, respectively, of fibril formation. Our results suggest a mechanism for self-assembly that includes the stabilization of transient α-helices through the formation of NMR-invisible helical intermediates followed by an α-helix to β-sheet conformational rearrangement. Furthermore, our results suggest that reducing intermolecular helix-helix contacts as in the S20P peptide is an attractive strategy for the design of blockers of peptide self-assembly.

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 phenylalanineglycine 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 (NPC). Here, we used NMR and isothermal titration calorimetry (ITC) to thermodynamically characterize these multivalent interactions. These analyses revealed that a combination of low per-FG motif affinity and the enthalpy-entropy balance prevents highavidity interaction between FG Nups and TFs, while 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 NPC and further expands our understanding of the mechanisms of "fuzzy" interactions involving IDPs.Intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs), constitute ~30-40% of the human proteome and are involved in many protein signaling and regulation processes (1). IDPs/IDRs can interact with their targets with high specificity, and yet often with low affinity and high reversibility. There is a broad interest in quantifying the thermodynamic driving forces governing IDP interactions. Many IDPs undergo a disorder-to-order transition upon binding to their targets (2), while others form 'fuzzy complexes' (3) where significant residual disorder is maintained in the interacting state. Due to their essential role in many biological processes, a better understanding of the energetics of IDP interactions is needed (4).Many IDP interactions are mediated by short linear motifs (SLiMs) that engage with receptor molecules. Because SLiMs do not have extensive interaction interfaces to induce high enthalpy, SLiM-containing IDPs often utilize multiple motifs to participate in multivalent interactions Thermodynamics of FG-Transport Factor Interaction2 enhances individually weak monovalent interactions, resulting in higher overall affinity (avidity) and specificity (6,7). One example of an IDR that utilizes multiple short linear motifs are disordered domains of Phe-Gly nucleoporins (FG Nups) which line the central channel of the nuclear pore complex (NPC) ( Figure 1A). FG Nups typically contain 5-50 FG motifs separated by spacer residues (8). These FG repeat regions collectively form a selectively permeable barrier for macromolecular transport through the NPC. Specific cargoes can translocate rapidly and efficiently through the NPC by binding to cognate transport factors (TFs). TFs make contacts with multiple FG repeat motifs, allowing them to diffuse rapidl...