A "grappling hook" interaction connects self-assembly and chaperone activity of Nucleophosmin 1

Abstract

How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers and undergoes liquid-liquid phase separation by binding RNA or ribosomal proteins. It provides the scaffold for ribosome assembly but also prevents protein aggregation as part of the cellular stress response. Here, we use aggregation assays and native mass spectrometry (MS) to examine the relationship between the self-assembly and chaperone activity of NPM1. We find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning-based structure prediction and cryo-electron microscopy reveal fuzzy interactions between the acidic disordered region and the C-terminal nucleotide-binding domain, which cross-link NPM1 pentamers into partially disordered oligomers. The addition of basic peptides results in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. Together, our findings show that NPM1 uses a "grappling hook" mechanism to form a network-like structure that traps aggregation-prone proteins. Nucleolar proteins and RNAs simultaneously modulate the association strength and chaperone activity, suggesting a mechanism by which nucleolar composition regulates the chaperone activity of NPM1.

Keywords: amyloid formation; membraneless organelles; molecular chaperones; native mass spectrometry.

Fig. 1.

NPM1 delays Aβ₄₂ aggregation. (a) NPM1 has a modular architecture, composed of a folded N-terminal pentamerization domain (NTD, residues 1 to 120), an intrinsically disordered region (IDR, residues 120 to 240) containing three acidic and two basic tracts (A1-3 and B1 and 2, respectively), and a C-terminal nucleotide-binding domain (CTD, residues 240 to 294). The NTD and the CTD are rendered based on PDB IDs 2P1B and 2VXD, respectively. (b) NPM1 pentamers associate with RNA, basic transcription factors, and ribosomal proteins to form the granular component of the nucleolus. Cellular stress induces the formation of nucleolar amyloid bodies. (c) The presence of increasing amounts of NPM1 shows that NPM1 delays the onset and the elongation of fibrillation, as judged by ThT fluorescence. Error bars indicate the SD of n = 4 experiments. (d) Truncated variants of NPM1 used in this study: NPM1₁₂₀ encompasses the NTD. NPM1₁₈₈ the NTD and the acidic regions of the IDR, NPM1₂₄₀ the NTD and the entire IDR and NPM1_240-294 only the CTD. (e) Fibrillation half-times (t _1/2) of Aβ₄₂ in the presence of 0 to 3 μM of NPM1 variants shows that only FL NPM1 and NPM1_240-294, but no other truncated variants, affects fibrillation. The t _1/2 of Aβ₄₂ alone is shown as a dashed line. Error bars indicate the SD of n = 4 experiments. A one-way ANOVA was used to test for the overall significant difference between groups before running pairwise t-tests with Bonferroni correction. Asterisks indicate significant differences (P < 0.001). (f) ThT fluorescence curves of Aβ₄₂ in the presence of 0 to 3 μM NPM1_240-294 show a dose-dependent delay in fibrillation and a decrease in fluorescence intensity. Error bars indicate the SD of n = 4 experiments.

Fig. 2.

Native MS shows that higher oligomerization is specific for NPM1_FL. (a) A schematic illustration of the native MS experiment, using collision-induced dissociation in the collision cell of the MS instrument. The illustration has been adapted from (43). (b) The native mass spectra of NPM1 at low collision energy show unresolved baseline humps, indicating large oligomers (top). Increasing the collision voltage releases NPM1 monomers, dimers, trimers, and pentamers (bottom). (c) The mass spectra of NPM1₁₂₀ show a range of oligomeric states composed of multiples of five subunits, as indicated. NPM1₁₈₈ and NPM1₂₄₀ form predominantly pentamers and minor decamer populations. (d) The isolated CTD (NPM1_240-294) exists nearly exclusively as monomers. The respective collision voltage at which each spectrum was obtained is indicated on the right.

Fig. 3.

An NTD–CTD interaction in FL NPM1. (a) The fluorescence of tryptophane residues 288 and 290 in NPM_240-294 is quenched by the addition of equimolar amounts of DNA or NPM₂₄₀, and to a lesser extent by NPM₁₂₀. In the presence of DNA and NPM₂₄₀, intermediate quenching is observed. Error bars indicate the SD of n = 6 repeats. One-way ANOVA was used to test for the overall significant difference between groups before running pairwiset-tests with Bonferroni correction. Asterisk indicates significance (** P < 0.01 and *** P < 0.001). (b) The cryo-EM reconstruction of the NTD (gray) shows virtually no deviations from the crystal structure (PDB ID 5EHD, blue), except for the A1 tract (residues 34 to 39), which could not be modeled based on the density map. (c) The cryo-EM density map for FL NPM1 reveals an additional asymmetric density (green) above the NTD pentamer (gray). (d) A second 3D class with fewer neighboring particles does not show the additional density. (e) The helical A2 tract (orange) appears as a diffuse density in the EM map. AF predicts a complex between the helical A2 tract (orange) and the basic CTD (rendered as an electrostatic surface). (f) Native MS of the NPM_ΔIDR variant lacking the disordered region between residues 120 and 240 reveals pentamers and a small fraction of decamers but no higher oligomers.

Fig. 4.

Modulating NPM1 self-assembly impacts its chaperone activity. (a) Residues 21 to 37 of rpL5 contain two basic motifs (B1 and B2). The native mass spectra of NPM1₁₂₀ show peak broadening and a shift to higher oligomeric states in the presence of rpL5_21-37. (b) FL NPM1 assemblies formed with rpL5_21-37 cannot be dissociated by collisional activation, as judged by the low abundance of monomers and the absence of dimers, trimers, or pentamers in the mass spectra. (c) Comparing the t _1/2 for fibril formation as judged by ThT fluorescence shows that rpL5_21-37 alone does not affect Aβ₄₂ aggregation, whereas rpL5_21-37 reduces the ability of NPM1 to delay Aβ₄₂ fibrillation. Error bars indicate the SD of n = 4 repeats. Significance was calculated using a Student's t-test for paired samples with equal variance. (d) Proposed connection between chaperone activity and self-assembly of NPM1. Under normal conditions, NPM1 (green) and basic proteins (blue) such as rpL5 form a tight nucleolar network. Under stress, rpL5 is released from the nucleolus, loosening up the NPM1 network, which enables NPM1 to sequester and chaperone amyloidogenic client proteins (gray). (e) Oligomerization and chaperones. Chaperone stoichiometries range from monomers (HSP70) to polydisperse oligomers (α-crystallin B). Chaperone activity of NPM1, on the other hand, involves the formation of large assemblies without defined stoichiometry.