Amyloid-like interactions within nucleoporin FG hydrogels

Abstract

The 62 kDa FG repeat domain of the nucleoporin Nsp1p forms a hydrogel-based, sieve-like permeability barrier that excludes inert macromolecules but allows rapid entry of nuclear transport receptors (NTRs). We found that the N-terminal part of this domain, which is characterized by Asn-rich inter-FG spacers, forms a tough hydrogel. The C-terminal part comprises charged inter-FG spacers, shows low gelation propensity on its own, but binds the N-terminal part and passivates the FG hydrogel against nonselective interactions. It was previously shown that a hydrophobic collapse involving Phe residues is required for FG hydrogel formation. Using solid-state NMR spectroscopy, we now identified two additional types of intragel interactions, namely, transient hydrophobic interactions between Phe and methyl side chains as well as intermolecular beta-sheets between the Asn-rich spacer regions. The latter appear to be the kinetically most stable structures within the FG hydrogel. They are also a central feature of neuronal inclusions formed by Asn/Gln-rich amyloid and prion proteins. The cohesive properties of FG repeats and the Asn/Gln-rich domain from the yeast prion Sup35p appear indeed so similar to each other that these two modules interact in trans. Our data, therefore, suggest a fully unexpected cellular function of such interchain beta-structures in maintaining the permeability barrier of nuclear pores. They provide an explanation for how contacts between FG repeats might gain the kinetic stability to suppress passive fluxes through nuclear pores and yet allow rapid NTR passage.

Fig. 1.

The Nsp1 FG/FxFG domain contains rigid and mobile segments. (A), (B) Investigation of rigid and mobile segments of the Nsp1 hydrogel using 2D (¹³C-¹³C) ssNMR employing through-space (A) and through-bond (B) mixing. Amino acid-specific assignments are indicated. (C) Relative (rel.) Cα-Cβ peak intensities per residue obtained from both spectra allow one to estimate the amino acid distribution within the rigid (left, blue) and mobile (right, red) segments of the hydrogel (n.d.: no Cα-Cβ peak detected). (D) Linear representations illustrating the nonuniform distribution of the various FG repeat types (Top) and relevant amino acids (Bottom) within the domain. (E) Bar models of Nsp1p fragments with high and low gel-forming propensity as determined by qualitative gelation assays (Table S1).

Fig. 2.

NMR characterization of Nsp1 hydrogel structure and gelation. (A) Details of NOESY spectra obtained for indicated Nsp1p fragments (250 ms mixing time) showing interresidue cross-peaks between indicated side chain methyls and Phe side chains. (B) Bar graph of C′, Cα, Cβ, and Hα chemical-shift differences for resonances of rigid Asn, Thr, and Ser of as compared to statistical average values for β-strands (green lines). (C) Dependence of Cα peak intensities for Asn, Thr, and Ser on the ¹H-¹H mixing time (t_HH) in NHHC (25) experiments suggests NH-HCα proton-proton distances compatible with β-strands. (D) NHHC spectrum obtained for a hydrogel containing ¹³C-labeled and ¹⁵N-labeled at a 1∶1 ratio. The signal arises due to short ¹H-¹H distances between complementary labeled β-strands forming an intermolecular sheet (see sketch and Materials and Methods for details).

Fig. 3.

, , and the N/Q-rich Sup35 prion domain specifically interact with Nsp1-derived hydrogels. (A) Hydrogel droplets containing 3 mM of the repeat domain were incubated with 2 μM of the indicated Atto488-labeled species (0.5 μM for Sup35^2-140). After 4 h, partitioning of the fluorescent species between buffer and gel phases was analyzed. Note that the full-length repeat domain (Nsp1^2-601) and the N-terminal portion with the highest gel-forming propensity (Nsp1^2-175) interacted strongly with the hydrogel. The prion domain of Sup35p (Sup35^2-140) showed an even higher partitioning into the gel phase. In comparison to a corresponding mutant repeat domain lacking all hydrophobic residues (Nsp1^274-601 Φ→S) or a 20 kDa PEG polymer, also was considerably enriched within the hydrogel. (B) Hydrogels with 3 mM were analyzed exactly as in (A). In comparison to the hydrogel, the inclusion of the module into the gel not only blocked binding sites for fluorescent molecules (Middle), but also efficiently suppressed influx and partitioning of the inert PEG control polymer into the gel (Lower).

Fig. 4.

The charged FSFG repeat domain facilitates entry of NTR•cargo complexes and passivates the hydrogel against nonspecific binding of inert proteins. Hydrogels consisting of 3 mM of the indicated repeat domains were formed as in Fig. 3. Influx of MBP-mCherry or an IBB-MBP-mEGFP•scImpβ complex into these hydrogels was analyzed after 90 sec, 10 min, and 30 min. Note that the intragel movement of IBB-MBP-mEGFP•scImpβ within the hydrogel was about 3-fold slower than in the hydrogel, while the enrichment of the NTR●cargo complexes at the buffer/gel boundary was increased by a factor of 2. The presence of the more C-terminal FxFG repeats harboring charged spacers also significantly reduced nonspecific binding of inert proteins to the hydrogels.

Fig. 5.

Comparison of the time-dependent decrease of the effective diffusion coefficient D_eff extracted from the PFG experiments (▴, logarithmic scale) and of the buildup of cross-polarization signals reflecting β-strands (○). Lines are drawn to guide the eye. The cartoon at the top illustrates the initial formation of protein clusters and the appearance of rigid β-strands. The bottom cartoon depicts formation of the gel (black) as followed by visual inspection of (as used for NMR).

Fig. 6.

Illustration of how an NTR might catalyze its own passage through an NQ-rich inter-FG repeat contact. (A) Drawing of an NPC filled with a schematized FG hydrogel. (B) Interrepeat contacts comprise rigid intermolecular β-sheets between NQTS-rich spacers (blue) and essential hydrophobic interactions between FG motifs (orange). The sum of interactions confers kinetic stability to these contacts, despite rapid fluctuations within the hydrophobic interactions. The energy barrier for dissociating such interrepeat contact should be lower when elementary interactions are broken not all at once but successively and if intermediates are stabilized by NTR-binding. (C–G) Binding of an NTR to FG motifs destabilizes and eventually dissociates the adjacent β-sheets, allowing the NTR to pass. The mesh contact is closed by the reverse reaction. Drawing is not to scale.