Franco A , Arranz R , Fernández-Rivero N , Velázquez-Campoy A , Martín-Benito J , Segura J , Prado A , Valpuesta JM , Muga A .

	Figure 1. Nucleoplasmin (NP) assembles the histone octamer. (A) Egg NP (eNP; from X. laevis) was incubated with either H2A-H2B or H3-H4 and the formed eNP/H2A-H2B or eNP/H3-H4 complexes were titrated with H3-H4 (B) and H2A-H2B (C), respectively. Samples were analyzed by 4–16% native PAGE and stained with Coomassie Brilliant Blue. The complex formed by two NP pentamers and a histone octamer is denoted with a black asterisk, and is also formed when both dimers were added at once (gel image from A). The complexes of NP with either H2A-H2B or H3-H4 dimers are marked with an orange asterisk. Inset: crystal structure (pdb 1K5J) of a monomeric NP core domain.
	Figure 2. Phosphorylation and exposure of the acidic tract regulates nucleoplasmin (NP)/histone octamer complex formation. (A) Schematic representation of the recombinant NP (rNP) variants used in this study. The phosphorylation sites (green circles) of egg NP (eNP; from X. laevis), the position of the acidic tracts in both protein domains (A1–3), and the location of the nuclear localization sequence (NLS) are shown. (B) Titration of native histone octamers (top panel) and crosslinked octamers (XL octamer, bottom panel) with eNP. Samples were analyzed by 4–16% native PAGE and stained with Coomassie Brilliant Blue. The complex formed by two NP pentamers and a histone octamer is marked with an asterisk. (C) Quantification of the NP/octamer complex marked with an asterisk in (B). The 440 kDa complex fraction was estimated as the ratio of the intensity of the NP/octamer complex band and that of eNP (squares), rNP (circles) or rNPΔ150–200 (triangles) in the absence of histones. Solid lines connecting the symbols have no physical meaning and are just to guide the eye. (D) Fluorescence spectra of the histone octamer (H2BT71C-Alexa 488) in the absence (black) and presence of eNP (green) or rNPΔ150–200 (blue). (E) Titration of the histone octamer (H2BT71C-Alexa488) with eNP (squares) or rNPΔ150–200 (circles). Histone concentration was 5 nM and NP concentration is given for the pentamer. Data are shown as mean ± s.d. of at least three different experiments. Data for rNPΔ150–200 have been shifted in the y-axis for the sake of clarity.
	Figure 3. CryoEM structure of the egg nucleoplasmin (eNP)/octamer complex. (A) Three orthogonal views of the eNP/octamer complex; bar = 100 Å. (B) The same three views with docking of the core domain of NP (pdb 1K5J; colored in red) in the two extremes of the complex, and the histone octamer (extracted from the structure of the nucleosome; pdb 1AOI), in the center of the 3D reconstruction. The two copies of H2A are colored in green, H2B in light blue, H3 in magenta and H4 in gold. The asterisks highlight the tails of the pentameric chaperone that interact with the base of the octamer. The other three tails are spread along the rest of the octamer structure. The reliability of the docking is depicted in Fig. S5, where the components of the complex are fitted into a map at a 4σ threshold.
	Figure 4. Cooperation of several nucleoplasmin (NP) C-terminal domains is required to bind the histone octamer. (A) H2A-H2B was incubated with increasing amounts of rNP113–200-4D for 1 h, samples were analyzed by 8% native PAGE and stained with Coomassie Brilliant Blue. (B) H2A-H2B/rNP113–200-4D complexes were crosslinked with bis(sulfosuccinimidyl)suberate (BS3), analyzed by denaturing NuPAGE Novex 4–12% Bis-Tris gel in MES buffer and stained with Coomassie. Bands are labeled with the stoichiometry inferred from their apparent molecular weight and their theoretical molecular weight in brackets. (C) The interaction between rNP113–200-4D and native or crosslinked histone octamers was studied by 8% native PAGE as in (A). (D) Saturation stoichiometries were estimated as the molar ratio of tail domain/histone H2A-H2B dimer (orange circles) or tail domain/crosslinked octamer (purple squares) at the inflexion point, using experimental data from (A) and (C). Data are presented as mean ± s.d. of at least three independent experiments; complexes of rNP113–200-4D with H2A-H2B or crosslinked octamer are designated with orange and purple asterisks, respectively, while black asterisks mark protein aggregates.
	Figure 5. Interaction between the nucleoplasmin (NP) C-terminal domain and histones does not induce structural rearrangements in any of these proteins. Far-UV CD spectra of isolated rNP113–200-4D and H2A-H2B dimers (A) or crosslinked histone octamers (C), and of the complex between the chaperone domain and the histones. The spectra of the complexes were compared with the theoretical spectrum, obtained upon addition of the spectra of each protein component measured independently (dashed lines). The ellipticity of each protein component and of the complexes generated upon their mixing was monitored at 222 nm as a function of temperature (from 20 to 90 °C), to study the effect of rNP113–200-4D /H2A-H2B (B) and rNP113–200-4D/crosslinked octamer (D) complex formation on their thermal stability. For the sake of comparison, the same protein concentration (10 µM for rNP113–200-4D, 10 µM for H2A-H2B and 2 µM for the histone octamer) was used in all samples. Molar ellipticity could not be employed, as one of the samples is a protein mixture. The measurements were done in 10 mM K/K2PO4 pH 7.5, 150 mM NaF with a protein concentration of 10 µM for H2A-H2B and rNP113–200-4D and 2 µM for the histone octamer.
	Figure 6. Nucleoplasmin (NP)-mediated transfer of histone octamers to DNA depends on chaperone post-translational modification and its C-terminal domain. 0.4 µM native (A) or crosslinked (B) histone octamers were incubated with increasing eNP concentrations for 1 h before the addition of 0.4 µM DNA. After 2 h at room temperature and 30 min at 42 °C, samples were analyzed by 6% native PAGE and stained with SRBR Green. The NP/Octamer molar ratio is noted above each lane. Bands marked with NS correspond to nucleosomes, and those with a single or double asterisk may be due to histone/DNA aggregates or to histone/DNA complexes generated upon transfer of crosslinked histone complexes. (C,D) NP-mediated nucleosome assembly was estimated as the intensity ratio of the nucleosome band in the presence of a given NP concentration and absence of the chaperone. Estimates are given for eNP (squares), rNP (circles; Fig. S6A,B) and rNPΔ150–200 (triangles; Fig. S6C,D), using native (C) or crosslinked (D) histone octamers. Lines connecting the symbols are just to guide the eye. Data are shown as mean ± s.d. of at least three independent experiments.
	Figure 7. Model for nucleoplasmin (NP)/histone octamer complex formation and chaperone-mediated octamer transfer to DNA. (A) NP can either bind sequentially the histone octamer components, assembling an octamer-like particle, or interact with preformed, stable histone octamers. The complex involves interaction of two NP pentamers and a histone octamer. In the presence of DNA, the NP-bound octamer is transferred to DNA, leading to nucleosome formation. (B) Comparison of the octamer structure (pdb 1AOI) docked into the CryoEM structure of the eNP/octamer complex and in the nucleosome. Note that the DNA strands are located in the same regions of the chaperone tails. The histone octamer employs common interacting surfaces to bind DNA or NP, offering a rationale for the competition between NP and DNA for octamer binding.

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